September 2018 Dates 22SAT 23SUN · Award Session 24 ... screen-saver, virus scan and power ... ※Time would be delayed due to traffic jam

Yoshihiko Saito, MD. (Professor, Department of Cardiovascular Medicine, Nara Medical University)Congress Chairman

NARA, JAPAN

Venue

Dates

2018September SAT SUN22 23

The 2nd JCS Council Forum on Basic CardioVascular Research

Program and Abstract

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Nara Kasugano International Forum IRAKA

Contents

Welcome Greetings � �� 3General Information � �� 4Information for Participants�� 5Information for Chairpersons and Speakers (Oral Presentations)�� 6Information for Chairpersons and Speakers (Poster Presentations)�� 7Access�� 8Floor Map� �� 11Poster Area�� 12Program at a Glance� �� 13

ProgramKeynote Lecture �� 16Sponsored Symposium �� 17Grant Session for Basic Research �� 21Luncheon Seminar �� 22Award Session �� 24Poster Presentation �� 25

AbstractsKeynote Lecture �� 44Sponsored Symposium �� 47Grant Session for Basic Research �� 71Luncheon Seminar �� 75Award Session �� 79Poster Presentation �� 83

Index�� 120Sponsors List / Acknowledgment �� 122


3

Welcome Greetings

Dear colleagues,

It is our great pleasure and honor to welcome you all to the 2nd JCS Council forum on Basic CardioVascular Research (BCVR) in Nara. The JCS Council on BCVR was established last year to promote the basic cardiovascular research and to encourage especially young scientists. Basic research is essential to understand the mechanism of cardiovascular diseases and develop new therapy.

The mission of the forum is to gain new knowledge and techniques in this research field and to exchange valuable information among investigators from many countries. We will have 2 keynote lectures by Prof. Walter Koch, Temple University School of Medicine, USA and Prof. Thomas Eschenhagen, University Medical Center Hamburg-Eppendorf, Germany, and 8 symposia by well-known outstanding speakers including investigators from overseas.

We received more than 140 abstracts for this forum. Several abstracts have been nominated as the candidates for the investigator awards, some of which will be presented in oral sessions and others will be considered for the poster awards. We hope to have good presentations and discussions.

All the participants along with their families are welcome to the get-together party in the Japanese Garden next to the conference hall on the first night of the forum. Award winners will be announced during this party.

As you know, Nara was the ancient capital of Japan for some time from AD 710. At those days, young Buddhist monks, young government officials, and young students went to Tang Dynasty, China, to learn Buddhism, new culture, new government system and new science, in spite of great danger and difficulty. Again, we are very happy to host the 2nd JCS Council Forum on BCVR here in Nara. We hope that this year’s forum will be a great success and we continued to have productive meetings for the years to come.

Please enjoy the meeting and your stay in the spectacular places that are rich in heritage in Nara.

With best regards,

Yoshihiko SaitoThe Congress Chairperson of the 2nd JCS council forum on BCVR

4

General InformationName of the Meeting / Congress Chairman：The 2nd JCS Council Forum on Basic CardioVascular ResearchYoshihiko Saito, MD.Department of Cardiovascular Medicine, Nara Medical University

Date：September 22 - 23, 2018

Venue：Nara Kasugano International Forum IRAKA101, Kasugano-cho, NaraTEL : +81-742-27-2630URL : http://www.i-ra-ka.jp/

Secretariat：c/o A & E Planning, Co., Ltd6th floor, Shin-Osaka Grand Bldg., 2-14-14, Miyahara, Yodogawa-ku, Osaka532-0003, JAPANTEL : +81-6-6350-7247E-mail：[email protected]

Official Website：https://www.aeplan.co.jp/bcvr2018/english/index.html


5

Information for ParticipantsRegistration DeskEntrance Hall, Nara Kasugano International Forum IRAKASeptember 22, 10 : 00 - 17 : 00 ／ September 23, 8:30 - 15:00

Registration Fee (on-site)Member : JPY 8,000Non-Member : JPY 10,000Undergraduate Students / International students with foreign nationalities residing in Japan / Graduate students residing outside of Japan / Graduate students who do not have medical doctor’s licenses and reside in Japan: Free (A valid Student ID card will be required at the registration desk.)

Name BadgesParticipants who completed the registration will receive name badges at the registration desk. Make sure to wear your name badge at all times during the forum.

Program and Abstract BookProgram and Abstract Book will be handed on the site.

CloakMeeting Room 2September 22, 10 : 00 -21 : 00September 23, 8 : 45 -16 : 00

Non-Smoking PolicySmoking is prohibited in the venue.

Photographing and Audio RecordingPhotographing and audio recording without permission are prohibited.

Mobile PhonePlease turn your mobile phones or and other electronic devices off or set them to silent mode before entering presentation rooms.

Dress CodeBusiness casual （no tie, no problem!）

Get-togetherDate : September 22, 19 : 00-21 : 00Fee : FreeVenue : Garden (In case of rain, it will be held at Reception Hall 2)*The Award winner(s) will be announced.

Luncheon SeminarTickets are not required. All participants are welcomed.Please directly come to the seminar rooms.

Photo SessionAll participants are requested to gather in Entrance Hall.Date : September 22, 16:40

6

Information for Chairpersons and Speakers (Oral Presentations)

For Chairpersons・ Please be seated at ‘Next Chairperson’s Seat’ in the front row in the session room at least 15 minutes

prior to your session.・Please start and end your session at the appointed time. Punctuality is appreciated.

For Speakers◦ Presentation Time

Special Lecture : as informed in advanceSponsored Symposium : as informed in advanceAward Session : 10 min. for presentation, 5 min. for discussion. Grant Session for Basic Research : 10 min. for presentation, 5 min. for discussion.

◦ Presentation InstrumentPresentations are restricted to Power Point Presentations using your own computers in the Oral sessions.

◦ PC PreviewPlease bring your computers to the PC Preview Desk (located at front-left side of the presentation room) 30 minutes prior to your presentation to check the output.

◦ Preparation of Presentation Data　◆ Notes on preparation of presentation data

・ Please create MS Power Point presentation (Windows ver. 2010 or later) and use the Windows standard fonts.

・ Please prepare your presentation data with the slide resolution XGA (1,024 × 768). Using of higher resolution may result in various projection trouble.

・ Please put ‘Abstract Code’ and ‘Your Name’ at the beginning of your filename.・ If your presentation data is linked to other files (i.e. still or moving images, graphs,etc.), those linked

files should also be saved in the same folder, and the links to be checked beforehand.・ If there are any COIs to be disclosed, the oral presenters should include the COI disclosure slide in

their presentation data. Presenters shall disclose applicable COI status at the beginning of the slide presentation or on a slide following the slide showing the title of presentation and name of presenters. Please visit “COI Disclosure” page for more details.

　◆ Notes on Bringing own computers・ Bring the AC adaptor for your computer. ・ A D-sub 15 pin monitor connector is necessary to connect your laptop to the projector equipment

provided in the presentation rooms. If your computer has a special-format monitor output terminal such as HDMI or Mini Display, bring a D- sub 15 pin conversion adaptor.

・ Deactivate password lock, screen-saver, virus scan and power-saving mode in advance. ・ Bring backup data of your presentation. We kindly ask you to have backups of your laptop content

to protect data from accidental loss.

Presentation Language and DataAll presentations must be delivered in English and all presentation data should be prepared in English.


7

Information for Chairpersons and Speakers (Poster Presentations)For Chairpersons・ Please come to the ‘Chairperson’s Check-in’ at 15 minutes prior to

your session and be ready in the poster session room at least 5 minutes prior to your session.

・ Please start and end your session at the appointed time. Punctuality is appreciated.

For SpeakersPoster Mounting, Discussion and RemovalThe secretariat will prepare a panel with your poster number. Tacks for putting up posters will be prepared at each panel.- Poster Mounting area is H160cm × W120cm.- Please prepare a slip with title, name(s) of author(s), and affiliation(s) with the size of H20cm × W100cm.

　[Mounting Date & Time] September 22, 15 : 30 - 16 : 00　[Removal Date & Time] September 23, 15 : 40 - 16 : 00

*Any posters left behind after the removal time shall be removed and discarded by the Secretariat. Posters still on display after the removal time has passed will be disposed by the Secretariat and will not be kept nor returned.

Poster SessionPoster presenters are requested to be ready in front of your poster at least 5 minutes prior to their presentation.　[Presentation Time]

September 22, 18 : 00 - 19 : 00Allotted time (one presenter) : 7 min.

Presentation Language and DataAll presentations must be delivered in English and all presentation data should be prepared in English.

Conflict of Interest DisclosureIf there is any COI, the poster presenter should use the disclosure format for the last of the presentation page, in order to clarify the names of companies and/or associations in question.For details, please refer to the official website. If there are any COIs to be disclosed, the poster presenters should include the COI disclosure in their posters. Presenters shall disclose applicable COI status at the end of their posters. Please visit “COI Disclosure” page for more details.

Poster AwardThe poster awards will be selected. The winner(s) will be announced at the Get-together.

8

Access

Port Liner:20min

KintetsuRapid Express:

30min

Hanshin Line:40min

Airport Limousine Bus: 75min

KintetsuRapid Express:

30minAirport Limousine Bus:

30min

Kintetsu Rapid Express: 30min

Nankai Rapid: 40min

JR: 5min

Yamatoji Rapid: 30minLimited Express Haruka:

30min

Airport Limousine Bus: 90min

Overnight Express Bus: 7.5hrs

JR Nara

Kintetsu-NaraYamato-Saidaiji

Kintetsu line / JR Nara

Kintetsu Line: 5minKintetsu Limited Express: 30min

Miyakoji Rapid: 43minShinkansen:

2.5hrs

FromTokyo

Tokyo Kyoto

Shinjuku

Shinkansen

JR

Kintetsu Line

Express Bus

FromAirports

TennojiKansai

InternationalAirport

OsakaItami

Airport

KobeAirport

Namba

JR Nara

Kintetsu-NaraYamato-Saidaiji


Other Railways

Hanshin LineTsuruhashi

Kintetsu Line: 5min

Kintetsu-NaraYamato-Saidaiji Osaka-

Uehommachi

Kintetsu Line: 5min

Nankai line

Kintetsuline


Kintetsu-NaraYamato-Saidaiji OsakaNamba

Kintetsu Line: 5minSannomiya

Kobe-Sannomiya

Airport Limousine Bus

JR

Kintetsu Line


9

[Walk]　20 minutes from Exit 2, Kintetsu Nara Station[Shuttle Bus Service]

We provide a free shuttle bus service to participants. Operating section: between Nara Kasugano International Forum IRAKA (the meeting venue) and JR Nara Station/Kintetsu Nara StationOperating dates: September 22-23

⬆to Kyoto

⬆to Kyoto

Nara Pref.Cultural Hall

Nara Pref.Museum of Art

Todaiji

Kasuga　 Taisha ➡Shrine　

Todaiji-Daibutsuden/Kasugataisha-mae

Bus Stop

Nara Kasugano InternationalForum Iraka-mae

Bus StopNara National

Museum

to Tenri⬇

Kofukuji

Nara Pref. Office

JRNara Sta.

　 ⬅Saidaiji　

Kintetsu Nara Sta.

Nara Kasugano International Forum IRAKA

Time Table September 22

［To the venue (I･RA･KA)］JR

NARA stationKintetsu

NARA station I･RA･KA

9:30 9:35 9:459:45 9:50 10:0010:00 10:05 10:1510:15 10:20 10:3510:30 10:35 10:5010:50 10:55 11:10

［From the venue (I･RA･KA)］

I･RA･KA Kintetsu NARA station

JR NARA station

19:30 19:40 19:5020:00 20:10 20:2020:30 20:40 20:5021:00 21:10 21:2021:15 21:25 21:3521:30 21:40 21:50

※ Time would be delayed due to traffic jam.

September 23［To the venue (I･RA･KA)］

JR NARA station

Kintetsu NARA station I･RA･KA

7:45 7:50 8:008:00 8:05 8:158:15 8:20 8:308:30 8:35 8:458:45 8:50 9:009:00 9:05 9:15

［From the venue (I･RA･KA)］

I･RA･KA Kintetsu NARA station

JR NARA station

15:40 15:50 16:0016:00 16:10 16:2016:20 16:30 16:4016:40 16:50 17:00

※ Time would be delayed due to traffic jam.

10

[Local Bus] Boarding points Kintetsu Nara Stn.: take buses at Nara Kotsu Bus Station No.1 near Exit 5JR Nara Stn. : take buses at Nara Kotsu East Bus Station No. 2

Bus lines and exiting points・Buses for “Kasuga Taisha Shrine”: exit at “Nara Kasugano International Forum Iraka-mae”・ Nara City Loop Line : exit at “Todaiji-Daibutsuden/Kasugataisha-mae”, then walk Daibutsuden

crossing for 3 minutes to the east.


11

Floor MapNara Kasugano International Forum IRAKA

Void

Hall Office

Elevator

Elevator

ConferenceRoom 1 & 2

ConferenceRoom 3 & 4

GurdenEntrance

1F

Restrant

YAMAHA CF

2F

ExhibionBooth

Meeting Room1-2

Room2

Noh TheatreRoom1

Reception Hall1Room 4

Room3Meeting Room

3-4

MeetingRoom 1

Meeting Room2Cloak

Meeting Room4Secretariat

MeetingRoom 3

SpecialMeetingRoom

Exhibion Booth

Books

DrawingRoom

DrinkService

12

Poster AreaReception Hall (2nd Floor, Nara Kasugano International Forum IRAKA)

P01-1

P01-8

P02-1 P03-8

P03-1

P05-1

P06-1

P17-1 P17-7 P18-1 P18-7

P07-1

P08-1

P09-1P06-8

P07-8

P10-1

P09-8

P10-8

P13-1

P12-8

P13-8

P16-1

P15-8

P16-7

P08-8

P11-1

P12-1

P11-8

P14-1

P15-1

P14-8P05-8

P04-8

P04-1

P02-8


13

Program at a Glance

September 23 (Sun.)Room 1

（Noh Theatre）Room 2

（Conference Room 1・2）Room 3


(Reception Hall 1)

9:009:00～10:30

Sponsored Symposium 5Genetics/Epigenetics

9:00～10:30

Sponsored Symposium 6Vascular�Biology10:00

10:30～11:45

Grant Session for Basic Research

11:00

12:0012:00～13:00

Luncheon Seminar 4

12:00～13:00

Luncheon Seminar 5

13:0013:10～14:00

Keynote Lecture 2Thomas�

Eschenhagen14:00

14:00～15:30

Sponsored Symposium 7Pulmonary�Hypertension/Vasculitis

14:00～15:30

Sponsored Symposium 8Arrhythmia

15:00

Closing�Remarks

16:00

17:00

18:00

19:00

20:00

21:00

September 22 (Sat.)Room 1

（Noh Theatre）Room 2



(Reception Hall 1)

Registration

Opening�Remarks11:00～12:30

Sponsored Symposium 1Heart�Failure

11:00～12:30

Sponsored Symposium 2Development�

andRegeneration

12:45～13:45

Luncheon Seminar 1

12:45～13:45

Luncheon Seminar 2

12:45～13:45

Luncheon Seminar 3

13:55～15:10

Award session

15:10～16:40

Sponsored Symposium 3Mitochondria�and�Cardiac�Metabolism

15:10～16:40

Sponsored Symposium 4Interorgan�

Communication�Network

Photo�Session17:00～17:50

Keynote Lecture 1

Walter�J.�Koch

18:00～19:00

Poster Presentation

19:00～21:00

Get-together/ Awards Ceremony

14

Poster Presentation

September 22, 18:00-19:0018:00

19:00

AgingChair person: Ichiro ShiojimaP01-1 ～ P01-8P.�25/P.�84

AortopathyChair person: Yasuhiro MaejimaP02-1 ～ P02-8P.�26/P.�86

Atherosclerosis/Thromboembolism1Chair person: Tetsuya MatobaP03-1 ～ P03-8P.�27/P.�88

Atherosclerosis/Thromboembolism2Chair person: Koji Ohashi P04-1 ～ P04-8P.�28/P.�90

Cardiac Hypertrophy and Function1Chair person: Hisakazu OgitaP05-1 ～ P05-8P.�29/P.�92

Cardiac Hypertrophy and Function2Chair person: Noboru FujinoP06-1 ～ P06-8P.�30/P.�94

Development and Differentiation/RegenerationChair person: Yoshinori YoshidaP07-1 ～ P07-8P.�31/P.�96

Drug Discovery/Translational ResearchChair person: Hironori NakagamiP08-1 ～ P08-8P.�32/P.�97

Genetics/CardiomyopathyChair person: Seiko OhnoP09-1 ～ P09-8P.�33/P.�99

Inflammation and FibrosisChair person: Yasuchika TakeishiP10-1 ～ P10-8P.�34/P.�101

Ischemic Cardiovascular DiseaseChair person: Eiki TakimotoP11-1 ～ P11-8P.�35/P.�103

Kidney/Interorgan Communication NetworkChair person: Toshihisa AnzaiP12-1 ～ P12-8P.�36/P.�105

Metabolic SyndromeChair person: Satoaki MatobaP13-1 ～ P13-8P.�37/P.�107

Pulmonary HypertensionChair person: Koichiro SugimuraP14-1 ～ P14-8P.�38/P.�109

Vascular BiologyChair person: Jun K. YamashitaP15-1 ～ P15-8P.�39/P.�111

ArrhythmiaChair person: Shigeki KobayashiP16-1 ～ P16-7P.�40/P.�113

Diabetes and CVDChair person: Yasuko K BandoP17-1 ～ P17-7P.�41/P.�115

Mitochondria/OrganellesChair person: Osamu YamaguchP18-1 ～ P18-7P.�42/P.�117

15


PROGRAM

16

Keynote Lecture

Keynote Lecture 1� September�22　17:00-17:50��Room��1（Noh�Theatre）　

Chairperson：Yoshihiko�Saito（Department of Cardiovascular Medicine, Nara Medical University）Sponsored�by��Takeda�Pharmaceutical�Company�Limited.

KL-1 　Targeting GRK2 in the Failing HeartWalter J Koch（ Lewis Katz School of Medicine, Temple University）

Keynote Lecture 2� September�23　13:10-14:00　Room�1（Noh�Theatre）

Chairperson：Issei�Komuro（ Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo）

KL-2 　3D Heart muscles from induced pluripotent stem cells for drug screening, disease modelling and cardiac repairThomas Eschenhagen（ University Medical Center Hamburg Eppendorf）


17

Sponsored Symposium 1 � September�22��11:00-12:30��Room�1（Noh�Theatre）

Heart FailureChairperson：Masafumi�Yano（ Faculty of Medicine and Health Sciences, Yamaguchi University）� Koichiro�Kuwahara（ Shinshu University School of Medicine）Sponsored�by��OTSUKA�Pharmaceutical�Co.Ltd.

SS1-1 　PP2A/HSP70 dynamically regulates HDAC2 S394 phosphorylation and its activity in cardiac hypertrophyHyun Kook（ Chonnam National University Medical School）

SS1-2 　Effects of sodium/glucose cotransporter 1 inhibitor on hypertrophic cardiomyopathy.Masamichi Hirose（ Iwate Medical University）

SS1-3 　Epigenomic regulation of cardiac remodeling during heart failureKoichiro Kuwahara（ Shinshu University School of Medicine）

SS1-4 　Protective role of intercalated disk protein afadin in the pressure-overloaded heartHisakazu Ogita（ Shiga University of Medical Science）

Sponsored Symposium 2 � September�22��11:00-12:30��Room�2（Conference�Room�1・2）

Development and RegenerationChairperson：Keiichi�Fukuda（ Keio University School of Medicine）� Hyo-soo�Kim（ Seoul National University）Sponsored�by��Astellas�Pharma�Inc./Amgen�Astellas�BioPharma�K.K.

SS2-1 　New cell surface receptor that specifies cardiac lineage commitment during differentiation from iPSCs and heart development in mouse and humanHyo-Soo Kim（ Strategic Center of Cell and Bio Therapy for Heart, Diabetes & Cancer /

Cardiovascular Center, Seoul National University Hospital, Seoul, Republic of Korea）

SS2-2 　Myocardial Tissue Engineering for Regenerative Therapy and Drug ScreeningTatsuya Shimizu（ Institute of Advanced Biomedical Engineering and Science, Tokyo

Women's Medical University）

SS2-3 　Safe and Effective Cardiac Regenerative Medicine with Human iPSCsShugo Tohyama（ Keio University School of Medicine）

SS2-4 　Generation of a Pacemaker Cells from Human Induced Pluripotent Stem Cells: Progress and IssuesJunya Aoyama（ Nippon Medical School Hospital）

Sponsored Symposium

18

Sponsored Symposium 3 � September�22��15:10-16:40��Room�1（Noh�Theatre）��

Mitochondria and Cardiac MetabolismChairperson：Tetsuji�Miura（ Sapporo Medical University School of Medicine）� Tomomi�Ide（ Faculty of Medical Sciences, Kyushu University）Sponsored�by��AstraZeneca�K.K.�/�ONO�PHARMACEUTICAL�CO.,�LTD.

SS3-1 　Mitochondrial fusion and fission proteins as targets for cardioprotectionDerek Hausenloy（ Duke-National University Singapore）

SS3-2 　Mass spectrometry-based omics approach reveals multifaceted roles of mitochondrial metabolism in the drug target, muscle differentiation, and maintaining its own DNA.Daiki Setoyama（ Clinical Laboratories, Kyushu University Hospital）

SS3-3 　Essential components to induce PINK1-Parkin mediated mitophagyAtsushi Hoshino（ Kyoto Prefectural University of Medicine）

SS3-4 　The critical roles of coagulation factors in inducing brown adipose tissue dysfunction in obesity.Yuka Hayashi（ Department of CardioVascular Biology and Medicine Niigata University

Graduate School of Medical and Dental Sciences）

Sponsored Symposium 4 � September�22��15:10-16:40�Room�2（Conference�Room�1・2）

Interorgan Communication NetworkChairperson：Thomas�N.�Sato（ Advanced Telecommunications Research Institute International）� Ichiro�Manabe（ Graduate School of Medicine, Chiba University）Sponsored�by��MSD�K.K.

SS4-1 　Senescence, the endothelium and a vascular protective geneJennifer R Gamble（ Centenary Institute, Sydney）

SS4-2 　Organ specific gene regulation by distinct vascular mechanisms during development.Thomas N Sato（ The Thomas N Sato BioBEC-X Laboratories, Advanced Telecommunications

Research Institute International (ATR), Kyoto, Japan ／ ERATO Sato Live Bio-Forecasting Project, Japan Science and Technology Agency (JST), Kyoto, Japan ／ Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA ／ Centenary Institute, Sydney, Australia）

SS4-3 　Immunometabolism in macrophage as a novel therapeutic target of metabolic syndromeYumiko Oishi（ Nippon Medical School）

SS4-4 　Specific Neural Stimulations Modulate the Formation of Immune Cell Gateways into the CNSDaisuke Kamimura（ Molecular Psychoimmunology, Institute for Genetic Medicine,

Hokkaido University）


19

Sponsored Symposium 5 � September�23��9:00-10:30��Room�1（Noh�Theatre）　

Genetics/EpigeneticsChairperson：Seiji�Takashima（ Graduate School of Medicine, Osaka University）� Koh�Ono（ Graduate School of Medicine, Kyoto University）

SS5-1 　Developing new Treatments for CardiomyopathyJianming Jiang（ National University of Singapore）

SS5-2 　Genetic analysis of more than 100K Japanese subjects including cardiovascular disordersYoichiro Kamatani（ Center for Genomic Medicine, Kyoto University Graduate School of

Medicine）

SS5-3 　Practice and Application of Whole Exome Sequencing in Hereditary Heart DiseaseYoshihiro Asano（ Osaka University Graduate School of Medicine）

SS5-4 　Single-nucleus RNA-seq analysis reveals pathogenic transcriptional programs in human heart failure.Kanna Fujita（ The University of Tokyo, Graduate School of Medicine, Cardiovascular

Medicine）

Sponsored Symposium 6 �September�23��9:00-10:30��Room�2（Conference�Room�1・2）　Vascular BiologyChairperson：Toyoaki�Murohara（ Nagoya University Graduate School of Medicine）� Yoshiaki�Kubota（ Keio University School of Medicine）Sponsored�by��Takeda�Pharmaceutical�Company�Limited.

SS6-1 　TIE receptor mediated signaling in lymphatic and blood vascular formation and maintenanceYulong He（ Soochow University）

SS6-2 　Impact of vascular endothelial stem cells on vascular integrityNobuyuki Takakura（ Department of Signal Transduction, Research Institute for

Microbial Diseases, Osaka University）

SS6-3 　The biological functions of thrombomodulin in endothelial cellsHua-Lin Wu（ Department of Biochemistry and Molecular Biology, College of Medicine,

National Cheng Kung University, Tainan, Taiwan.）

SS6-4 　Role of thrombospondin-1 in mechanotransduction and development of thoracic aortic aneurysmYoshito Yamashiro（ University of Tsukuba）

20

Sponsored Symposium 7 � September�23��14:00-15:30��Room�1（Noh�Theatre）　

Pulmonary Hypertension / VasculitisChairperson：Yoshikazu�Nakaoka（ National Cerebral and Cardiovascular Center Research Institute）� Noriaki�Emoto（ Kobe Pharmaceutical University）

SS7-1 　VEGFR3 as a novel modulator of BMP signaling in pulmonary hypertensionSuk-Won Jin（ Gwangju Institue of Science and Technology/Yale University）

SS7-2 　Inlerleukin-6 Blockade Therapy for Pulmonary Arterial Hypertension and Large Vessel VasculitisYoshikazu Nakaoka（ Department of Vascular Physiology, National Cerebral and

Cardiovascular Center Research Institute）

SS7-3 　Cell-cell Contact Promotes Endothelial Integrity by BMPR2 Mediated Metabolic and Epigenetic ChangesKazuya Miyagawa（ Kobe Pharmaceutical University）

SS7-4 　The single nucleotide polymorphism of MLX gene is involved in the development of Takayasu ArteritisYasuhiro Maejima（ Tokyo Medical and Dental University）

Sponsored Symposium 8� September�23��14:00-15:30��Room�2（Conference�Room�1・2）

ArrhythmiaChairperson：Tetsushi�Furukawa（ Medical Research Institute, Tokyo Medical and Dental University）� Katsushige�Ono（ Faculty of Medicine, Oita University）Sponsored�by��Bayer�Yakuhin,�Ltd

SS8-1 　Cardiovascularpathophysiology of fatty acid binding proteinsWei Wang（ Hebei Medical University）

SS8-2 　Redox Physiology of TRP channelsYasuo Mori（ Kyoto University Graduate School of Engineering）

SS8-3 　Extracellular nucleotides: Novel players for the pathogenesis of atrial fibrillationTetsuo Sasano（ Tokyo Medical and Dental University）

SS8-4 　Single Cell Analysis of CALM-related LQT iPS Cell model using membrane voltage imaging systemYuta Yamamoto（ Kyoto University）


21

Grant Session for Basic Research � September�23��10:30-11:45��Room�1�(Noh�Theatre)

Chairperson：Naoki�Mochizuki（ National Cerebral and Cardiovascular Center, Research Institute）� Keiichi�Fukuda（ Keio University School of Medicine）

GS-1 　Systematic comprehension of developmental origins of adult-onset cardiovascular diseasesYuichiro Arima（ Department of Cardiovascular Medicine）

GS-2 　New Insight into the Molecular Drug Target of Cardio-renal SyndromeTakuya Kumazawa（ Cardiovascular Medicine, Nara Medical University, Japan）

GS-3 　Development of in vivo four-dimensional [4D] metabolism imagingYoshinori Katsumata（ Keio University School of Medicine）

GS-4 　Mitochondria-nucleus network for a novel therapy against heart failureAtsushi Hoshino（ Department of Cardiovascular medicine, Kyoto Prefectural University

of Medicine）

GS-5 　Myocardial Regeneration by Engineering Mitochondrial TurnoverWataru Kimura（ RIKEN Center for Biosystems Dynamics Research）


22

Luncheon Seminar 1� September�22　12:45-13:45��Room�2�(Conference�Room�1・2)�

Chairperson：Michihiro�Yoshimura�（ Division of Cardiology, Department of Internal Medicine, The Jikei University School of Medicine）

Sponsored�by��Takeda�Pharmaceutical�Company�Limited.

LS1-1 　Environmental or Energetic Factors Regulating Mitochondrial Respiration.Norihiko Takeda（ Department of Cardiovascular Medicine. Graduate School of

Medicine, The University of Tokyo）

LS1-2 　Deep Learning-Based System for the Research of Pluripotent Stem Cell-derived cellsShinsuke Yuasa（ Department of Cardiology, Keio University School of Medicine）

Luncheon Seminar 2� September�22��12:45-13:45��Room�3�(Conference��Room�3・4)

Chairperson：Yasushi�Sakata�（Osaka University）Sponsored�by��Mitsubishi�Tanabe�Pharma�Corporation.

Takuya Kishi（ Kyushu University）

Luncheon Seminar 3� September�22　12:45-13:45��Room�4�(Reception�Hall�1)

Chairperson：Hiroyuki�Tsutsui�（Kyushu University）Sponsored�by��Bristol-Myers�Squibb�/�Pfizer�Japan�Inc.

LS3 　Onco-Cardiology: Exploring a New Frontier with Basic CardioVascular ResearchHiroshi Akazawa（ The University of Tokyo）

Luncheon Seminar 4� September�23��12:00-13:00��Room�2�(Conference�Room�1・2)

Chairperson：Issei�Komuro�（ Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo）

Sponsored�by��Daiichisankyo�Co.,�Ltd.

Shigeo Horinaka（ Department of Cardiology and Nephrology, Dokkyo Medical University）

LS2 　

LS4 　

Luncheon Seminar


23

Luncheon Seminar 5� ��September�23�12:00-13:00��Room�3�(Conference��Room�3・4)Chairperson：Tetsuji�Miura（Department of Cardiovascular, Renal and Metabolic Medicines）

Sponsored�by��Nippon�Boehringer�Ingelheim�Co.,�Ltd�and�Eli�Lilly�Japan�K.K.

LS5 　Understanding SGLT2 Inhibitors from the Perspective of Drug Development –The Clinical Possibilities of Pharmacological Characteristics and Mitochondrial Metabolism – Masashi Suganuma（Suganuma Clinic）

24

Award Session� September�22��13:55-15:10��Room�1�(Noh�Theatre)

Chairperson：Tetsuo�Minamino（Faculty of Medicine, Kagawa University）� Masaki�Ieda（Faculty of Medicine, University of Tsukuba）

AS-1 　Cell-free DNA Released in Atrial Fibrillation Promotes Systemic Pro-Inflammatory Cytokine ExpressionMasahiro Yamazoe（ Department of Bio-informational Pharmacology, Medical Research

Institute, Tokyo Medical and Dental University）

AS-2 　Increased G α o expression underlies cardiac dysfunction accompanied with abnormal Ca2+ handlingHideaki Inazumi（ Department of Cardiovascular Medicine, Kyoto University Graduate

School of Medicine）

AS-3 　Direct Reprogramming with Sendai Virus Vectors Improves Cardiac Function In Vitro and In VivoKazutaka Miyamoto（ Keio University School of Medicine）

AS-4 　Blockade of NKG2D / NKG2D Ligand Interaction Attenuated Post-infarct Myocardial InjuryKotaro Matsumoto（ Laboratory of Clinical Science and Biomedicine, Graduate School

of Pharmaceutical Sciences, Osaka University）

AS-5 　(Pro)renin receptor promotes development of sarcopenia via activation of Wnt- YAP signaling pathwayJin Endo（ Keio University School of Medicine）

Award Session


25

Poster Presentation 01� September�22��18:00-19:00��Room�4�(Reception�Hall�1)AgingChairperson：Ichiro�Shiojima（ Kansai Medical University）

P01-1 　Nucleolus-mediated Cellular Senescence is involved in Heart FailureTakuya Kumazawa（ Cardiovascular Medicine, Nara Medical University, Japan）

P01-2 　Amino acid substitution of Werner gene is responsible for cardiac agingTakahiro Kamihara（ Department of Cardiology, Nagoya University Graduate School of

Medicine）

P01-3 　Endothelial-SASP induces premature senescence in adipocytes and disrupts metabolic homeostasisAgian Jeffilano Barinda（ Clinical Pharmaceutical Laboratory, Kobe Pharmaceutical

University ／ Cardiovascular Medicine Division, Kobe University）

P01-4 　Endothelial cell senescence exacerbates heart failure due to pressure-overloadDhite B Nugroho（ Department of Internal Medicine, Division of Cardiovascular

Medicine, Graduate school of medicine, Kobe University ／Departement of Internal Medicine, Gadjah Mada University）

P01-5 　Repetitive Postprandial Combined Metabolic Spikes Induce Premature Aging of Bone MarrowKeita Horitani（ Department of Medicine II Kansai Medical University）

P01-6 　Visceral adipose tissue potentiates cardiac dysfunction in aging and obesityDaigo Sawaki（ INSERM U955, University of Paris East Créteil）

P01-7 　Osteoprotegerin as a protective factor in the aging heartToshihiro Tsuruda（ University of Miyazaki）

P01-8 　Endothelial cell senescence is involved in the progression of atherosclerosisSakiko Honda（ Department of Cardiovascular Medicine, Graduate School of Medical

Science, Kyoto Prefectural University of Medicine）

Poster Presentation

26

Poster Presentation 02� September�22��18:00-19:00��Room�4�(Reception�Hall�1)AortopathyChairperson：Yasuhiro�Maejima（ Tokyo Medical and Dental University）

P02-1 　Molecular bases for the sex difference in aortic aneurysm formation in Marfan syndromeQing Liu（ Graduate School of Medicine and Faculty of Medicine, The University of

Tokyo）

P02-2 　The integrity of extracellular matrix affects transduction of mechanical stress in the aortic wallThang Quoc Bui（ Department of Cardiovascular Surgery, University of Tsukuba）

P02-3 　Cell-specific function of fibulin-4 in progression of ascending aortic aneurysm in miceCaroline Antunes Lino（Department of Anatomy, University of Sao Paulo, Institute of Biomedical Sciences, Sao Paulo, Brazil）

P02-4 　Role of PAR1-Egr1 in the initiation of thoracic aortic aneurysmSeungjae Shin（ Tsukuba university）

P02-5 　The role of Syk in pathogenesis of aortic dissectionYouhei Hashimoto（ Department of Internal Medicine, Division of Cardiovascular

Medicine, Kurume University School of Medicine）

P02-6 　Involvement of Focal adhesion kinase in pathogenesis of aortic dissectionRyohei Majima（ Division of Cardiovascular Medicine, Department of Internal Medicine,

Kurume University School of Medicine）

P02-7 　Impact of CD44 on the development of thoracic aortic aneurysm and dissection in miceOmer Hatipoglu（ Okayama University）

P02-8 　Angiotensin receptor-Neprilysin Inhibition Attenuates Aortic Aneurysm in MiceKouhei Tashiro（ Fukuoka University School of Medicine）


27

Poster Presentation 03� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Atherosclerosis/Thromboembolism1Chairperson：Tetsuya�Matoba（ Department of Cardiovascular Medicine, Graduate School of Medical Sciences,

Kyushu University）

P03-1 　30-day morbidity and mortality of patients with acute coronary syndromeThatsaphone Somsaart（ Setthathirath Hospital）

P03-2 　STEMI PATIENT IN TYPE C HOSPITAL : TREAT OR NOT TO TREAT ?Lianita Gumdani（ Mekarsari Hospital）

P03-3 　Aortic valve calcification and coronary atherosclerosis in familial hypercholesterolemia patientsEri Yamamoto（ Department of Cardiology, Tokyo Women's Medical University）

P03-4 　Stabilization of symptomatic carotid atherosclerotic plaques by statins: A pathological analysisTakao Konishi（ Department of Cancer Pathology, Hokkaido University, Graduate School

of Medicine）

P03-5 　Validation of OCT findings in vivo plaque histology obtained by directional coronary atherectomyHiroshi Okamoto（ Kawasaki medical school）

P03-6 　Influence of thrombin on the structure of platelet glycoprotein Ib α binding to von Willebrand factorShinichi Goto（ Keio University School of Medicine ／ Tokai University School of

Medicine）

P03-7 　Different impact of thrombin generation rate and enzymatic effects of thrombin on thrombus growth.Shinichi Goto（ Keio University School of Medicine ／ Tokai University School of

Medicine）

P03-8 　Bacteroides inhibits atherosclerosis by regulating gut microbial LPS productionNaofumi Yoshida（ Kobe University Graduate School of Medicine）

28

Poster Presentation 04� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Atherosclerosis/Thromboembolism2Chairperson：Koji�Ohashi�（ Nagoya University Graduate School of Medicine）

P04-1 　Comprehensive analysis of HIF-1a target genes in macrophage adaptation to the microenvironmentTakayuki Isagawa（ Department of Cardiovascular Medicine, Nagasaki University

Graduate School of Biomedical Sciences）

P04-2 　Telomerase Deficiency Prevents Vascular Smooth Muscle Cell Proliferation and Neointima FormationJun Aono（ Department of Cardiology, Pulmonology, Hypertension, and Nephrology,

Ehime University Graduate School of Medicine）

P04-3 　Glucocorticoid decreases uptake of Ac-LDL through suppression of JAK-STAT pathway in macrophagesRyota Hashimoto（ Department of Physiology, Juntendo University Faculty of Medicine）

P04-4 　Direct interaction between adiponectin and oxidized LDL limits their action each otherAkemi Kakino（ Department of Physiology, Shinshu University School of Medicine）

P04-5 　DPP-4 inhibitors protect HUVECs from hypoxia-induced apoptosis and vascular barrier impairmentMasanori Hirose（ Department of Cardiovascular Medicine, Chiba University Graduate


P04-6 　Pericyte-specific deletion of Ninjurin1 induces abnormal vasa vasorum and intimal hyperplasia.Kiwamu Horiuchi（ Department of Cardiovascular Regeneration and Innovation,

Asahikawa medical university）

P04-7 　Metabolic Stress Evokes Mitochondrial Dysfunction and Vascular Senescence via Mitochondrial FissionYoshihiro Uchikado（ Cardiovascular Medicine and Hypertension, Graduate School of

Medicine, Kagoshima University）

P04-8 　Angiotensin Receptor Neprilysin Inhibitor Preserved Cardiac Function in Atherosclerotic MouseYasunori Suematsu（ Fukuoka University）


29

Poster Presentation 05� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Cardiac Hypertrophy and Function1Chairperson：Hisakazu�Ogita（ Shiga University of Medical Science）

P05-1 　Protein kinase N promotes cardiac hypertrophy and fibrosisTeruhiro Sakaguchi（ Nagoya university）

P05-2 　Cavin-2/SDPR Deficiency Attenuates Myofibroblast Differentiation and Cardiac Fibrosis in TAC MiceYusuke Higuchi（ Department of Cardiovascular Medicine, Kyoto Prefectural University

of Medicine）

P05-3 　Sirt7 plays a distinct role in cardiomyocyte and non-cardiomyocyte in response to pressure overloadSatoru Yamamura（ Kumamoto University）

P05-4 　The Potent p300-HAT Inhibitor GO-Y030 Suppresses TAC-induced Development of Heart Failure in miceKana Shimizu（ Division of Molecular Medicine, Graduate School of Integrated

Pharmaceutical and Nutritional Sciences, University of Shizuoka ／Clinical Research Institute, Kyoto Medical Center, National Hospital Organization）

P05-5 　Amlexanox or Dantrolene prevents angiotensin II-induced cardiac hypertrophy in neonatal myocytesTetsuro Oda（ Department of Medicine and Clinical Science, Division of Cardiology,

Yamaguchi University Graduate School of Medicine）

P05-6 　A ryanodine receptor stabilizer as a novel strategy for pressure-overloaded heart failureMichiaki Kohno（ Yamaguchi University）

P05-7 　Verification of novel molecular mechanism of cardiac hypertrophy via regulation of mRNA degradationYuki Masumura（ Department of Cardiovascular Medicine, Osaka University Graduate


P05-8 　A Novel Long Noncoding RNA Protects the Heart from Heart Failure Accompanied by Renal DysfunctionTakuya Kumazawa（ Cardiovascular Medicine, Nara Medical University, Japan）

30

Poster Presentation 06� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Cardiac Hypertrophy and Function2Chairperson：Noboru�Fujino（ Pharmaceutical and Health Sciences, Kanazawa University）

P06-1 　Analysis of molecular dynamics of TN-C in Kawasaki disease using a murine model induced by FK565Daiki Yamamoto（ Departmtent of Pathology and Matrix Biology, Mie University

Graduate School of Medicine）

P06-2 　Cocoa Bean Polyphenols Inhibit Pressure Overload-Induced Cardiac Hypertrophy and Heart FailureNurmila Sari（ Division of Molecular Medicine, Graduate School of Pharmaceutical

Sciences, University of Shizuoka）

P06-3 　Guanylyl Cyclase-A Signaling Attenuates the Deleterious Salt Effect on Mineralocorticoid ReceptorHitoshi Nakagawa（ Nara Medical University）

P06-4 　α 1A-adrenergic receptor suppresses cardiac hypertrophy in pregnancy-associated hypertensive miceChulwon Kwon（ Graduate school of Life and Environmental Sciences, University of

Tsukuba, Tsukuba, Ibaraki, Japan）

P06-5 　Left Ventricular Global Longitudinal Strain Values in Healthy Filipino Adults: Cross sectional StudyJulius Caesar de Vera（ St. Luke's Medical Center-Global City）

P06-6 　Right Ventricular Strain in Healthy Adult Filipinos A Retrospective Cross Sectional Pilot StudyJulius Caesar de Vera（ St. Luke's Medical Center-Global City）

P06-7 　Change of exhaled acetone concentration levels in patients with acute decompensated heart failureTetsuro Yokokawa（ Department of Cardiovascular Medicine, Fukushima Medical

University）


31

Poster Presentation 07� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Development and Differentiation/RegenerationChairperson：Yoshinori�Yoshida（ Center for iPS Cell Research and Application, Kyoto University）

P07-1 　Transcriptional regulation and physiological significance of ALK1 target genes in endothelial cellsOsamu Nakagawa（ Department of Molecular Physiology, National Cerebral and


P07-2 　Significance of Hey transcriptional factors in endothelial cells during cardiovascular developmentOsamu Nakagawa（ Department of Molecular Physiology, National Cerebral and


P07-3 　A novel origin of ECs; Isl1-positve cells contribute to caudal vessel formation in zebrafishHiroyuki Ishikawa（ National Cerebral and Cardiovascular Center Research Institute）

P07-4 　Identification of new myocardial growth factor and its application to cardiac regenerationSho Haginiwa（ Keio University）

P07-5 　tie1 but not tie2 is essential for cardiovascular development in zebrafishNanami Morooka（ National Cerebral and Cardiovascular Center）

P07-6 　Myocardial canonical Wnt signaling in zebrafish heart developmentAyano Chiba（ National Cerebral and Cardiovascular Center Research Institute）

P07-7 　Extended in vitro culture enhanced maturation and engraftment of human iPSC-derived cardiomyocytesShin Kadota（ Department of Regenerative Science and Medicine, Shinshu University ／

Institute for Biomedical Sciences, Shinshu University）

P07-8 　EFFICIENT CARDIAC REGENERATION THERAPY BY CELL CYCLE ACTIVATION OF IPSC-DERIVED CARDIOMYOCYTESMnabu Kasamoto（ Department of Cardiovascular Medicine, Kyoto University Graduate


32

Poster Presentation 08� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Drug Discovery/Translational ResearchChairperson：Hironori�Nakagami（ Department of Health Development and Medicine, Osaka University


P08-1 　Development of PCSK9 epitope vaccine for dyslipidemia in miceHironori Nakagami（ Osaka University）

P08-2 　Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug TestingHirohiko Kohjitani（ Department of Cardiovascular Medicine, Kyoto University Hospital）

P08-3 　Development of a new high-throughput screening assay for the discovery of novel heart failure drugsHikaru Moki（ Division of Molecular Medicine, School of Pharmaceutical Sciences,

University of Shizuoka, Shizuoka, Japan）

P08-4 　A bacteria-derived ACE2-like enzyme suppresses cardiac remodeling and dysfunction in mice.Takafumi Minato（ Akita University Graduate School of Medicine）

P08-5 　The effect of mTORC1 inhibition on necroptotic signaling-mediated suppression of autophagic fluxKoki Abe（ Sapporo Medical University, Department of Cardiovascular, Renal and

Metabolic Medicine）

P08-6 　Silencing of microRNA-34a ameliorates aortic valve calcification via Notch1-Runx2 signalingTaku Toshima（ Yamagata University School of Medicine）

P08-7 　A Novel Target Molecule of Nobiletin possess a Therapeutic Potency for Heart Failure in MiceYoichi Sunagawa（ School of Pharmaceutical Sciences, University of Shizuoka ／ Clinical

Research Institute, Kyoto Medical Center ／ Shizuoka General Hospital）

P08-8 　Heparin mobilizes human multipotent mesoangioblasts from the heart during cardiac catheterizationYoshihiro Hata（ Department of Medicine II, Kansai Medical University）


33

Poster Presentation 09� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Genetics/CardiomyopathyChairperson：Seiko�Ohno（ Department of Bioscience and Genetics, National Cerebral and Cardiovascular

Center）

P09-1 　Identification and arrhythmogenic significance of a novel TMEM168 mutation in Brugada syndromeDimitar Zankov（ Shiga University of Medical Science）

P09-2 　Overlapping Brugada syndrome associated with SCN5A mutations in patients with sick sinus syndromeAsami Kashiwa（ Department of Cardiovascular Medicine, Kyoto University）

P09-3 　The Ref/Alt imbalance of the VUS in RNAseq affect the gene expression in cardiomyopathyYohei Miyashita（ Osaka university Dept. of Cardiovascular Medicine ／ Osaka university

Dept. of Legal Medicine）

P09-4 　The validation of association of a rare variant in TKS5 with atrial fibrillation (AF) and examination of its role in AF pathogenesisXiaoxi Yang（ Bio-informational Pharmacology, Tokyo Medical and Dental University,

Japan）

P09-5 　Gene Expression Landscape of Heart Failure Mice model caused by Cardiac Specific PRMT1 DeficiencyWeizhe Lu（ Ph.D. Program in Human Biology, School of Integrative Global Majors (SIGMA),

University of Tsukuba）

P09-6 　Establishment of Fabry cardiomyopathy model and its isogenic control from a female patientYuki Kuramoto（ Department of Cardiovascular Medicine, Osaka University Graduate


P09-7 　Involvement of Attenuation of β-Adrenoreceptors Signaling in Development of Takotsubo SyndromeTomoya Nakano（ Department of Cardiovascular Medicine, Nara Medical University）

P09-8 　Single cell analysis of cardiomyocyte reveals spatial and temporal heterogeneity in heart failureMasahiro Satoh（ Department of Cardiovascular Medicine, Chiba University Graduate

School of Medicine ／ Genome Science Laboratory, Research Center for Advanced Science and Technology, The University of Tokyo）

34

Poster Presentation 10� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Inflammation and FibrosisChairperson：Yasuchika�Takeishi（ Department of Cardiovascular Medicine, Fukushima Medical University）

P10-1 　Maternal high-fat diet promotes the expansion of abdominal aortic aneurysm in adult offspring.Makoto Saburi（ Department of Cardiovascular Medicine Kyoto Prefectural University of

Medicine）

P10-2 　“Human” epicardial adipose tissue induces “rat” atrial myocardial fibrosis through paracrine effectsShintaro Kira（ Department of Cardiology and Clinical Examination, Oita University）

P10-3 　NLRP3 Inflammasome Initiated through CaMKII δ in Cardiomyocytes is Essential for Cardiac RemodelingTakeshi Suetomi（ Yamaguchi University ／ University of California San Diego）

P10-4 　Involvement of VGLL3 in TGF-beta-induced epithelial-to-mesenchymal transitionNoritaka Yamaguchi（ Department of Molecular Cardiovascular Pharmacology,

Graduate School of Pharmaceutical Sciences, Chiba University ／Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University）

P10-5 　MAIR-II deficiency prevents excessive inflammation and fibrosis post-myocardial infarctionSaori Yonebayashi（ Department of Cardiology, University of Tsukuba）

P10-6 　The role of cellular senescence in cardiac fibrosis after myocardial infarctionMasato Shibamoto（ Department of Cardiovascular Medicine,Osaka University


P10-7 　Identification of Cardiovascular Dysfunction in Spondyloarthritis Model RatsHiroki Hayashi（ Osaka University）

P10-8 　Beneficial effects of exercise on cardiac fibrosis in obese mice: the role of leptin.Maria Pini（ IMRB - Inserm U955）


35

Poster Presentation 11� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Ischemic Cardiovascular DiseaseChairperson：Eiki�Takimoto（ Department of Cardiovascular Medicine, Graduate School of Medicine, The

University of Tokyo）

P11-1 　Prevalence of risk factors for CVD among children and adolescents of Asian Indian originPartha Sarathi Datta（ Department of Anthropology, Visva-Bharati, Santiniketan, India.）

P11-2 　Akt1-mediated muscle growth promotes blood flow recovery by enhancing HO-1 in neighboring cellsYasuhiro Izumiya（ Osaka City University）

P11-3 　Iron Metabolism in a Mouse Model of Hind Limb IschemiaKeisuke Okuno（ Cardiovascular Division, Department of Internal Medicine, Hyogo

College of Medicine）

P11-4 　AIM Depletion Suppressed Cardiac Rupture without Affecting Hemodynamics after MI in MiceShohei Ishikawa（ Department of Cardiorenal and Cerebrovascular Medicine, Faculty of

Medicine, Kagawa University）

P11-5 　MURC/Cavin-4 deficiency improves cardiac ischemia-reperfusion injuryYumika Tsuji（ Department of Cardiovascular Medicine, Kyoto Prefectural University of

Medicine）

P11-6 　Lack of Sirt7 attenuates myocardial ischemia-reperfusion injury by modulating NRF2 activitySatoshi Araki（ Department of Cardiovascular Medicine, Faculty of Life Sciences,

Kumamoto University）

P11-7 　5-Aminolevulinic Acid Combined with Ferrous Iron Ameliorates Cardiac Ischemia/Reperfusion Injury.Yu Oimatsu（ Kumamoto University）

P11-8 　Human iPSC-derived cardiac tissue transplantation to a rat unloaded ischemic heart modelDaisuke Heima（ Department of Cardiovascular Surgery, Graduate school of Medicine,

Kyoto University, Kyoto, Japan ／ Department of Cell Growth and Differentiation, Center for IPS Cell Research and Application (CiRA), Kyoto university, Kyoto, Japan）

36

Poster Presentation 12� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Kidney/Interorgan Communication NetworkChairperson：Toshihisa�Anzai（ Department of Cardiovascular Medicine, Hokkaido University Graduate School

of Medicine）

P12-1 　The Body-wide Transcriptome Landscape of Disease ModelsKyoji Urayama（ The Thomas N. Sato BioMEC-X Laboratories, Advanced Telecommunications

Research Institute International (ATR), Kyoto, Japan ／ ERATO Sato Live Bio-Forecasting Project, Japan Science and Technology Agency (JST), Kyoto, Japan）

P12-2 　Purification of the growing skeletal muscle derived exosomal micro RNA.Toshifumi Ishida（ Kumamoto University）

P12-3 　Gut Microbiota Depletion Alters Cardiac Hypertrophy in MiceTakehiro Kamo（ The Institute for Adult Diseases, Asahi Life Foundation）

P12-4 　The alterations of gut epithelial barrier function in a mouse model of heart failureAkiko Saga-Kamo（ Department of Cardiovascular Medicine, The University of Tokyo

Hospital）

P12-5 　Gut microbiome composition and plasma microbial metabolites in patients with heart failureTomohiro Hayashi（ Kobe University Graduate School of Medicine）

P12-6 　Serum FABP4 is removed by the kidney via glomerular filtration and megalin-mediated reabsorptionTatsuya Iso（ Gunma University Graduate School of Medicine）

P12-7 　Transferrin receptor 1 is associated with the development of diabetic kidney diseaseSeiki Yasumura（ Cardiovascular Division, Department of Internal Medicine, Hyogo

College of Medicine）

P12-8 　Renal Tissue Hypoxia in the Subacute Phase of Renal Ischemia Reperfusion InjuryPei Chen Connie Ow（ National Cerebral and Cardiovascular Research Institute ／

Monash University）


37

Poster Presentation 13� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Metabolic SyndromeChairperson：Satoaki�Matoba（ Department of Cardiovascular Medicine, Graduate School of Medical Science,

Kyoto Prefectural University of Medicine）

P13-1 　Natriuretic Peptide Induces Adipose Tissue Browning and Thermogenesis in Diet Induced Obese MiceHaruka Kimura（ The Jikei University School of Medicine Division of Cardiology）

P13-2 　Brown adipose tissue dysfunction is involved in the pathologies in failing heartYohko Yoshida（ Niigata University）

P13-3 　Psychological stress accelerates HFD-induced insulin resistance by enhancing NE activity in eWATShinichiro Motoyama（ Kyoto Prefectural University of Medicine）

P13-4 　Adrenomedullin plays a compensatory role in obesity-related hypertensionKosai Cho（ Department of Primary Care and Emergency Medicine, Kyoto University


P13-5 　Maternal high-fat diet impairs insulin sensitivity by enhancing NLRP3 inflammasome activationNaotoshi Wada（ Kyoto Prefectural Univ of Med）

P13-6 　Loss of periostin ameliorates adipose tissue inflammation and fibrosis in vivoTakahiro Horie（ Department of Cardiovascular Medicine, Kyoto University Hospital）

P13-7 　Effects of Tofogliflozin on Cardiac Hypertrophy in Metabolic Model RatsTomonari Kimura（ Department of Cardiovascular Medicine, Okayama University

Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama, Japan.）

P13-8 　Insufficient neonatal ketogenesis triggers the susceptible trait for hepatic steatosisYuichiro Arima（ Department of Cardiovascular Medicine, Kumamoto University）

38

Poster Presentation 14� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Pulmonary HypertensionChairperson：Koichiro�Sugimura（ Department of Cardiovascular Medicine, Tohoku University Graduate School

of Medicine）

P14-1 　SOX17 mutations in Japanese patients with pulmonary arterial hypertensionTakahiro Hiraide（ Keio University School of Medicine）

P14-2 　Pristane might be useful for creating a novel model of CTD-PAH in miceHiroyoshi Mori（ National Cerebral and Cardiovascular Center Research Institute）

P14-3 　TrkB might have a role for right ventricular homeostasis under chronic hypoxia-induced PHMakoto Okazawa（ National Cerebral and Cardiovascular Center）

P14-4 　Hypoxia Causes Epigenetic Changes in SMCs of PAH with BMPR2 MutationKazufumi Nakamura（ Okayama University）

P14-5 　INHBA is a Novel Gene that Regulates Pulmonary Arterial Hypertension DevelopmentGusty Rizky Teguh Ryanto（ Division of Cardiovascular Medicine, Department of

Internal Medicine, Kobe University Graduate School of Medicine）

P14-6 　Role of endothelin-1 in the regulation of ALK-1 in human pulmonary arterial endothelial cellsRyo Kawamata（ Fukushima Medical University）

P14-7 　Expressional analysis of Endothelin-2 in Lung under Hypoxic ConditionDonytra Arby Wardhana（ Clinical Pharmacy, Kobe Pharmaceutical University）

P14-8 　Loss of Fam13a exacerbates the vascular remodeling in pulmonary artery hypertension.Yuko Kuribayashi（ Laboratory of Clinical Pharmaceutical Science, Kobe Pharmaceutical

University, Kobe, Japan）


39

Poster Presentation 15� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Vascular BiologyChairperson：Jun�K.�Yamashita（ Center for iPS Cell Research and Application (CiRA), Kyoto University）

P15-1 　The role of ORAIP for the coronary artery plaque instability in Familial HypercholesterolemiaHidekimi Nomura（ Tokyo Women's Medical University）

P15-2 　Phenotypic change of perivascular adipose tissue associated with endovascular damageYusuke Adachi（ Department of Cardiovascular Medicine, Graduate School of Medicine,

The University of Tokyo, Tokyo, Japan）

P15-3 　Adipose-ERK2 Protect From Ectopic Fat Depositions and Vascular Dysfunction in ObesityAyumu Osaki（ National Defense Medical College）

P15-4 　The role of redox regulation of PKG1 α on sympathetic nervous system in salt sensitive hypertension.Nobuyuki Tokunaga（ Department of Cardiovascular Medicine, Graduate School of

Medical Sciences, Kumamoto University, Kumamoto, Japan.）

P15-5 　ChGn-2 Deletion Reduces Lipoprotein Retention and PDGF-mediated Aortic Smooth Muscle Cells MigrationImam Manggalya Adhikara（ Division of Cardiovascular Medicine, Department of

Internal Medicine, Kobe University Graduate School of Medicine）

P15-6 　Cre expression in hematopoietic cells of smooth muscle cell-targeted Cre recombination miceTomohiko Ishibashi（ National Cerebral and Cardiovascular Center Research Institute）

P15-7 　SREBF1/MicroRNA-33b axis exhibits potent effect on unstable atherosclerotic plaque formation in vivoTakahiro Horie（ Kyoto University）

P15-8 　Unanticipated roles of IKK β in vascular calcificationNoboru Ashida（ Kyoto University Graduate School of Medicine, Department of

Cardiovascular Medicine）

40

Poster Presentation 16� September�22��18:00-19:00��Room�4�(Reception�Hall�1)ArrhythmiaChairperson：Shigeki�Kobayashi（ Yamaguchi University Graduate School of Medicine）

P16-1 　Atrial Fibrillation Detection Using Convolutional Neural NetworksXue Zhou（ University of Aizu）

P16-2 　Non-uniform calcium dynamics of the atrial cells and Purkinje fibers with references to T tubulesHideo Tanaka（ Kyoto Prefectural University of Medicine）

P16-3 　THREE-DIMENSIONAL STRUCTURAL ANALYSIS OF MUTANT RYR2 CHANNELS ASSOCIATED WITH CPVTJingshan Gao（ Department of Cardiovascular Medicine, Kyoto University Graduate


P16-4 　Glucose fluctuations in diabetic mice induce cardiac sinoatrial node dysfunction.Takahiro Oniki（ Department of Cardiology and Clinical Examination, Faculty of

Medicine, Oita University）

P16-5 　SERCA2 C674S heterozygote knock-in mice with angiotensin infusion leads to ventricular arrhythmiaKei Ito（ National Defense Medical College）

P16-6 　Inhibition of HDAC 6 manipulates electrophysiological properties of HL-1 cellsPeili Li（ Tottori University）

P16-7 　Pacemaker activity generated by CRISPR/Cas9 based genome editing for Kcnj2Kensuke Ihara（ Department of Bio-informational Pharmacology, Tokyo Medical and

Dental University）


41

Poster Presentation 17� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Diabetes and CVDChairperson：Yasuko�K�Bando（ Department of Cardiology, Nagoya University Graduate School of Medicine）

P17-1 　Mechanisms for Ca2+ signaling defects underlying diastolic dysfunction in diabetic cardiomyopathyYoshinori Mikami（ Department of Physiology, Faculty of Medicine, Toho University）

P17-2 　Physiological role of TRPC6 upregulation in hyperglycemic rodent heartsSayaka Oda（ Division of Cardiocirculatory Signaling, National Institute for Physiological

Sciences (Exploratory Research Center on Life and Living Systems), National Institutes of Natural Sciences ／ Department of Physiological Sciences, SOKENDAI (School of Life Science, The Graduate University for Advanced Studies)）

P17-3 　Activation of SERCA2 ameliorated endothelial function and skeletal muscle function of db/db miceToyokazu Kimura（ National Defense Medical College）

P17-4 　Understanding the pathogenesis of diastolic dysfunction: from the whole heart to the myofilamentMark T Waddingham（ National Cerebral and Cardiovascular Center, Suita, Osaka,

Japan）

P17-5 　Impact of insulin resistance on right heart failure in sugen chronic hypoxia ratsHuiling Jin（ Department of Cardiac Physiology National Cerebral Cardiovascular Center

Research Institute）

P17-6 　SGLT1 exhibits ischemic tolerance and contributes to cardioprotection in diet-induced obese miceAkira Yoshii（ The Jikei University School of Medicine, Division of Cardiology）

P17-7 　Glucagon plays protective role against catecholamine surge in heart failureKazuyuki Nishimura（ Nagoya University Graduate school of Medicine）

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Poster Presentation 18� September�22��18:00-19:00��Room�4�(Reception�Hall�1)Mitochondria/OrganellesChairperson：Osamu�Yamaguch（ Department of Cardiology, Pulmonology, Hypertension & Nephrology,

Ehime University Graduate School of Medicine）

P18-1 　Novel compound heterozygous TMEM70 variants impaired the activity of ATP synthase of complex VKeiichi Hirono（ University of Toyama）

P18-2 　HERPUD1 protects against ER stress- and oxidative stress-induced cardiomyocyte deathSoichiro Ikeda（ Department of Cardiovascular Medicine, Graduate School of Medical

Sciences, Kyushu University, Fukuoka, Japan）

P18-3 　FKBP8 protects the heart by preventing accumulation of misfolded protein and ER-associated apoptosisTomofumi Misaka（ Fukushima Medical University）

P18-4 　Oxidant stress terminates protective signals in mitochondria by recruitment of DUSP5 and PHLPP-1.Wataru Ohwada（ Sapporo Medical University ／ JCHO Sapporo Hokushin Hospital）

P18-5 　Inhibition of xanthine oxidase prevents skeletal muscle mitochondrial dysfunction in heart failureHideo Nambu（ Department of Cardiovascular Medicine, Faculty of Medicine and

Graduate School of Medicine, Hokkaido University）

P18-6 　Cilnidipine improves heart failure after myocardial infarction by suppressing mitochondrial fissionKakeru Shimoda（ Creative Research Group on Cardiocirculatory Dynamism, Exploratory

Research Center on Life and Living Systems (ExCELLs), National Institutes of Natural Sciences ／ Department of Physiological Sciences, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI)）

P18-7 　Supplementation of succinyl-CoA improves the mitochondrial complex II dysfunction in heart failureSatoshi Maekawa（ Hokkaido University）

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Abstracts

44

Keynote Lecture

Abstract

Sponsored by Takeda Pharmaceutical Company Limited.


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Keynote Lecture 1 Room 1 （Noh Theatre） September 22　17:00-17:50　Chairperson：Yoshihiko�Saito（Department of Cardiovascular Medicine, Nara Medical University）

Kl-1 Targeting GRK2 in the Failing Heart

Walter�J�KochLewis Katz School of Medicine, Temple University

We have spent the last two decades investigating novel molecular targets for correcting ventricular dysfunction in heart failure. We have identified the G protein-coupled receptor (GPCR) kinase-2 (GRK2) as such a target. We have shown that inhibiting the activity of this kinase or genetic deleting this kinase in the heart can prevent and reverse heart failure in mouse models. Moreover, a gene therapy approach with a peptide inhibitor of GRK2 (βARKct) has been used in several small and large animal models to rescue heart failure. This includes a recent study in a pre-clinical pig model of heart failure and βARKct gene delivery reversed ventricular dysfunction and caused reverse remodeling. A clinical trial is being planned and these studies will be presented. In addition to a gene therapy approach for GRK2 inhibition we are pursuing small molecule pharmacological inhibition and recent studies have shown the FDA approved anti-depressant drug paroxetine is a specific GRK2 target outside its actions to prevent serotonin re-uptake. We have recently showed that paroxetine can reverse heart failure in an animal model and this therapeutic effect is independent to its CNS effects. This paves the way for paroxetine derivatives and other small molecules to target GRK2 for future heart failure therapy, which are being pursued. Thus, GRK2 inhibition is a viable novel and innovative treatment for heart failure. In addition to GRK2 being pathological chronically, we have also shown it can promote acute cell death after ischemic injury and oxidative stress. This is actually due to non-canonical activity of GRK2 in the mitochondria. Our latest results on understanding the role of mitochondrial GRK2 will also be presented.

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Kl-2 3D Heart muscles from induced pluripotent stem cells for drug screening, disease modelling and cardiac repair

Thomas�EschenhagenUniversity Medical Center Hamburg Eppendorf

The availability of principally unlimited quantities of human cardiomyocytes from induced pluripotent stem cells (iPSC) provide unprecedented opportunities for drug screening, disease modeling and regenerative therapies. While principally straightforward, challenges remain in terms of the genetic instability of iPSC cultures over time, the immaturity and functional variability of human iPSC-derived cardiomyocytes, the limited robustness of assays evaluating their phenotype, and the translatability of iPSC-based cardiac repair. Some of these shortcoming may be overcome by providing hiPSC cardiomyocytes with a more physiological 3D growth environment. 3D Heart muscle patches could also solve the low retention rate of hiPSC cardiomyocytes when injected directly into injured hearts. The lecture will give an overview of the current state and means to move the field forward with a focus on tissue engineering.

Keynote Lecture 2 Room 1 （Noh Theatre） September 23　13:10-14:00　Chairperson：Issei�Komuro（ Department of Cardiovascular Medicine, Graduate School of Medicine, The



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siumSponsored Symposium 1

Heart Failure

Abstract

Sponsored by OTSUKA Pharmaceutical Co.Ltd.

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SS1-2Effects of sodium/glucose cotransporter 1 inhibitor on hypertrophic cardiomyopathy.Masamichi Hirose1）,�Miho�Ibi1）,�Maki�Saito1）

１）Iwate Medical University

Accumulating basic and clinical evidence has been reported regarding the effectiveness of selective sodium-glucose co-transporter 2 (SGLT2) inhibitors to treat cardiovascular diseases and events. Several recent studies demonstrated that: 1) cardiac overexpression of SGLT1 causes cardiac hypertrophy and increases interstitial fibrosis in transgenic mice, and 2) the increased gene expression of SGLT1 was associated with human hypertrophic and ischemic cardiomyopathy. We also demonstrated that cardiac hypertrophic cardiomyopathy induced by chronic pressure overload was improved in SGLT1 knockout mice, suggesting that SGLT1 is a new therapeutic target for hypertension-induced cardiomyopathy. However, the effects of the selective SGLT1 inhibitor on hypertrophic cardiomyopathy are still unknown. Therefore, we investigated the effects of KGA-2727, a selective SGLT1 blocker, on pressure overload-induced hypertrophic cardiomyopathy. C57BL/6J male mice (10 or 11 weeks old) were subjected to transverse aortic constriction (TAC) and implanted with osmotic minipumps (Alzet,2006) containing KGA-2727 (100 mg/ml) or DMSO (as a control) subcutaneously. Six weeks after KGA-2727 infusion started, KGA-2727 reduced the increased heart weight, and improved the reduction of left ventricular fractional shortening in TAC-operated mice. In addition, KGA-2727 reduced the increased gene expressions of connective tissue growth factor (CTGF) and collagen type I in TAC-operated mouse ventricles. Moreover, KGA-2727 reduced the increased interstitial fibrosis in TAC-operated mouse ventricles. In contrast, KGA-2727 did not affect the serum glucose concentration regardless of TAC operation. In vitro, KGA-2727 suppressed phenylephrine-induced mouse neonatal cardiomyocyte hypertrophy. Our results suggest that a selective SGLT1 blocker such as KGA-2727 may be effective for the improvement of hypertrophic cardiomyopathy.

Sponsored Symposium 1: Heart Failure Room 1 （Noh Theatre） September 22　11:00-12:30　Chairperson：Masafumi�Yano（ Faculty of Medicine and Health Sciences, Yamaguchi University）� Koichiro�Kuwahara（ Shinshu University School of Medicine）SS1-1PP2A/HSP70 dynamically regulates HDAC2 S394 phosphorylation and its activity in cardiac hypertrophyHyun Kook1）

１）Chonnam National University Medical SchoolCardiac hypertrophy represents a form of global remodeling in response to increased hemodynamic demands. Previously we have reported that inhibition of class I HDAC can block the cardiac hypertrophy (Kee et al., 2006) and that histone deacetylase (HDAC) 2 plays a crucial role in the process (Kee et al., 2008). Recently, we also demonstrated that acetylation of K75 (Eom et al., 2014) and following casein kinease 2α1 (CK1α1)-induced phosphorylation of S394 (Eom et al., 2011) are critical for the activation of HDAC2 and hypertrophy. However, the detailed mechanism for modulating HDAC2 phosphorylation remains unclear. We identified the complex of HDAC2 and protein phosphatase (PP) 2A and heat shock protein 70 (Hsp70) were confirmed. Hypertrophic stresses led Ppp2ca to dissociate from Hdac2, which then recruited CK2α1 to phosphorylate Hdac2. Forced expression of Ppp2ca negatively regulated the hypertrophic response but PP2A inhibitor itself provoked hypertrophy. Phosphor-mimicking mutant of Hdac2 successfully reversed the anti-hypertrophy effect of Ppp2ca, which implicated that Ppp2ca targeted Hdac2 S394 phosphorylation. Hsp70 preferentially bound to phosphorylated Hdac2. Increased expression of Hsp70 led Ppp2ca to dissociate from Hdac2. Hsp70 inhibitor, Pifithrin-μ, significantly alleviated both cardiac hypertrophy and fibrosis induced by pressure overload. Transgenic mice expressing Ppp2ca negatively regulated isoproterenol-induced cardiac hypertrophy. However, double transgenic mice expressing both Ppp2ca and Hsp70 showed cardiac hypertrophy. Taken together, our results suggest that HDAC2 forms a complex with PP2A in the absence of hypertrophic stresses and remains inactivated. HDAC2 activation requires both detachment of PP2A and recruitment of CK2α1 for phosphorylation, which is maintained by the association with HSP70 during development of cardiac hypertrophy.


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　SS1-3Epigenomic regulation of cardiac remodeling during heart failureKoichiro Kuwahara1）

１）Shinshu University School of Medicine

Genetic reprogramming participates in the cardiac remodeling by affecting myocardial cellular function and survival. We revealed several molecular pathways that regulate epigenomic alterations in cardiac gene transcription, which is involved in the pathological cardiac remodeling and heart failure. A transcriptional repressor NRSF regulates multiple fetal cardiac genes expression. Inhibition of NRSF by over expression of dominant-negative form of NRSF or genetic deletion of NRSF in the heart resulted in cardiac dysfunction and sudden arrhythmic death accompanied with re-expression of multiple fetal cardiac genes. We demonstrated that fetal type cardiac ion channels, T-type Ca2+ channels and HCN channels, play important roles in the process leading to sudden arrhythmic death, concomitantly with activation of renin-angiotensin system that participates in generating arrhythmogenic substrates, and sympathetic nervous system. Furthermore recently we have found that GNAO1 is a potential NRSF target gene that plays a critical role in the reduction of cardiac contractile function. NRSF-GNAO1 pathway seems to regulate Ca2+ handling, thereby maintaining normal cardiac contractile function. We also revealed the functional significance of TRPC6 ion channel in calcineurin-NFAT signaling pathways that play critical roles in pathological cardiac remodeling. Indeed, inhibition of TRPC significantly ameliorated the pathological cardiac hypertrophy in mice. We are now addressing the possible therapeutic implication of TRPC inhibition for cardiovascular diseases. Collectively, our studies unraveled the transcriptional pathways involved in the development of pathological cardiac remodeling leading to heart failure and proposed potential therapeutic targets for the treatment and prevention of heart failure.

　SS1-4Protective role of intercalated disk protein afadin in the pressure-overloaded heartHisakazu Ogita1)

1）Shiga University of Medical Science

Afadin is expressed ubiquitously and an actin-binding scaffold protein. In the heart, it localizes at the intercalated disk to support cell adhesion molecules that connect neighboring cardiomyocytes. Afadin is critical in the embryonic development, because afadin knockout mice showed embryonic lethality. Thus, the role of afadin in each organ including the heart remains largely unknown. To explore its role, we newly generated cardiomyocyte-specific afadin conditional knockout (afadin cKO) mice. Gross appearance and cardiac phenotype of afadin cKO mice were indistinguishable from the control littermates in the physiological conditions. Then, pressure overload by transverse aortic constriction (TAC) was applied to the mice. The pressure gradient across the TAC site was 41 mmHg, which was evaluated by pulse-wave Doppler mode of ultrasonography. After TAC procedure, left ventricular (LV) thickness was increased in both control and afadin cKO mice, but at 8 weeks after TAC, the thickness was thinner in afadin cKO mice than control mice. More remarkably, LV ejection fraction was reduced in afadin cKO mice. Cardiac fibrosis, infiltration of inflammatory cells, and apoptosis were significantly increased in the afadin cKO heart, compared with the control heart. At the molecular level, afadin interacted with the cytoplasmic region of transforming growth factor (TGF)-β receptor, and the interaction is essential for the regulation of signaling transduction downstream of TGF-β receptor, resulting in the protection against the TAC-induced pressure overload-mediated cardiac insult. Finally, pharmacological inhibition of TGF-β receptor function mimicked the phenotypes observed in the absence of afadin. These results suggest that afadin exerts the protective role in the pressure-overloaded heart together with TGF-β receptor.

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Sponsored Symposium 2Development and Regeneration

Abstract

Sponsored by Astellas Pharma Inc.

Amgen Astellas BioPharma K.K.


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　SS2-2Myocardial Tissue Engineering for Regenerative Therapy and Drug ScreeningTatsuya Shimizu1）

１）Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University

Recent advances in stem cell biology and tissue engineering are realizing regenerative therapy and human tissue model fabrication. As one of the tissue engineering technologies, we have developed “cell sheet-based tissue engineering” which utilize cell sheets harvested from temperature-responsive culture dishes. In contrast to cell injection therapy, cell sheet transplantation improves cell survival, resulting in better therapeutic potential. Myoblast sheet transplantation has been already clinically applied for severe heart failure. It has been considered that main mechanism of heart function improvement is paracrine effects. That is, cytokines secreted from myoblast sheets promote blood vessel formation and inhibit cardiomyocyte apoptosis and tissue fibrosis, which lead to improvement of heart function. Next generation of regenerative therapy for severe heart failure is transplantation of three-dimensional (3-D) beating cardiac tissues, which might support heart function mechanically. We have already succeeded in fabricating 3-D beating human cardiac tissues by stacking human iPS cell-derived cardiomyocyte sheets. For scaling up, perfusable micro capillaries have been successfully introduced in 3-D cardiac tissues by using endothelial co-culture, perfusion bioreactor system and multi-step cell sheet layering process. Transplantation of human vascularized cardiac tissues into porcine models is now on going. On the other hand, we have also applied engineered beating human cardiac tissues to drug screening model. Macroscopically beating cardiac tissues has enabled direct measurement of contraction force. When the constructs are stretched, contraction force increased, which was consistent with Fran k-Starling Law. Several anti-cancer drugs decreased the contraction force as seen in clinical practice. These human myocardial tissues should be useful as a new evaluation system for cardiotoxicity and cardio pharmacology. Thus, cell sheet-based myocardial tissue engineering has great potential to engineer functional 3D cardiac tissues which might contribute to future regenerative therapy and drug screening.

Sponsored Symposium 2: Development and Regeneration Room 2 (Conference Room 1・2) September 22　11:00-12:30　Chairperson：Keiichi�Fukuda�（ Keio University School of Medicine）� Hyo-soo�Kim（ Seoul National University）SS2-1New cell surface receptor that specifies cardiac lineage commitment during differentiation from iPSCs and heart development in mouse and humanHyo-Soo Kim1）

１）Strategic Center of Cell and Bio Therapy for Heart, Diabetes & Cancer / Cardiovascular Center, Seoul National University Hospital, Seoul, Republic of Korea

Identifying lineage-specific markers is pivotal for understanding developmental processes and developing cell therapies. Here, we report a new cardiac-specific surface marker, latrophilin 2 (LPHN2), expressed specifically by cardiac progenitor cells and cardiomyocytes (CMCs) during mouse and human pluripotent stem cell (PSC) differentiation in vitro and exclusively in the heart during mouse embryonic development. Lphn2 knockout in mice is embryonically lethal because of severe heart, but not vascular, defects. Analysis of the signaling pathway indicated that cyclin-dependent kinase 5 is downstream of LPHN2 and collaborates with Src kinase to induce P38MAP kinase phosphorylation, subsequently activating cardiac-related gene transcription. PSC-derived LPHN2+ cells, but not LPHN2- cells, differentiated into CMCs and regenerated the myocardium when transplanted into infarcted hearts. Transplanted LPHN2+ cells improved left ventricle systolic function and reduced infarct sizes. These findings provide a valuable tool for isolating cardiomyogenic progenitors and CMCs from PSCs and shed light on heart development and regeneration.

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　SS2-4Generation of a Pacemaker Cells from Human Induced Pluripotent Stem Cells: Progress and IssuesJunya Aoyama1),�Kohei�Homma2),�Nari�Tanabe3),�Yasuo�Miyagi1),�Takashi�Nitta1)

1）Nippon Medical School Hospital, 2）Keio University School of Medicine, 3）Suwa University of Science

ObjectivePacemaker cells derived from human induced pluripotent stem cells (hiPSCs) could be applied to the next-generation therapy. However, it is unclear whether hiPSCs derived pacemaker could functionally stimulates the heart tissue for a long period. The purpose of this study was to induce the pacemaker cells and cardiomyocytes differentiation from hiPSCs, those are labeled with fluorescent proteins by using genome editing, and analyze the stimulating and contracting function, the protein and the mRNA expression in these cells at each maturation step.MethodsTo generate hiPSCs knock-in cell lines, CRISPR/Cas9 genome editing technique was applied. We generated two cell lines to detect HCN4 expression for pacemaker cells and αMHC expression for cardiomyocytes, respectively. The cells formed a spherical structure and were put into a bioreactor, with some cytokines. The differentiated beating spherical-structures were analyzed by a video analyzing system, immunohistochemistry, calcium imaging, and qRT-PCR at each day of differentiation.ResultsThe established knock-in cell lines were cultured to form spherical-structures. They differentiated and showed HCN4 expression with YFP-positive response or α MHC expression with GFP-positive response. The differentiated spherical-structures, began to beat, the beating rhythm being irregular and fast in the early differentiation stage, and regular and slower in the late of the differentiation stage. Specific cardiac proteins were detected by immunohistochemistry (Troponin T, MYH6, α -Actinin, HCN4, and Connexin43). Especially, connexin43 expression gradually increased and the expression patterns were changed from dot-shaped to linear, surrounding the cell as they matured. Calcium imaging showed the synchronization of the beating as the expression level of the connexin43 became higher. The expression levels of HCN4, Tbx3, Tbx18, and Nkx2-5, each related to pacemaker cell differentiation, also changed as they matured; especially, HCN4 expression was significantly decreased. ConclusionsThe levels of expressed proteins and mRNA in the derived pacemaker cells, including cardiomyocytes, changed as they matured. The beating rhythm also became slower and synchronized. In future, we plan to identify other gene networks related to the generation of pacemaker cells.

　SS2-3Safe and Effective Cardiac Regenerative Medicine with Human iPSCsShugo Tohyama1）,�Jun�Fujita1）,�Hideaki�Kanazawa1）,�Keiichi�Fukuda1）

１）Keio University School of Medicine

Cardiac regenerative therapy using human induced pluripotent stem cells (hiPSCs) is a potentially promising strategy for patients with heart disease; however, the inability to eliminate residual hiPSCs and generate a massive amount of pure hiPSC-derived cardiomyocytes (hiPSC-CMs) has been a barrier to realizing this potential. Recently, we established an innovative method for purifying the bulk of hiPSC- CMs by focusing on glucose, glutamine and lactate metabolism in hiPSCs and differentiated cardiomyocytes (Cell Stem Cell 2013, Cell Metabolism 2016, Circ. Res. 2017). In addition, we also developed an advanced two-dimensional culture system using multilayer culture plates with active gas ventilation that yielded a large number of pure hiPSC- CMs (Stem Cell Reports 2017). However, there are no efficient massive hiPSC-CM spheroids production and transplantation systems for heart failure patients. Thus, we developed a large-scale production system for pure hiPSC-CM spheroids using microwell culture plates on a clinically relevant scale, and an injection device for the direct intramyocardial hiPSC-CM spheroids transplantation (in revision). An injection device yielded an optimal three-dimensional distribution of transplanted hiPSC-CM spheroids in the porcine myocardial layer. These findings will provide a foundation for the clinical application of pure hiPSC-CM spheroids-mediated safer cardiac regenerative therapy for heart failure patients.


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Mitochondria and Cardiomyocyte Metabolism

Abstract

Sponsored by AstraZeneca K.K.

ONO PHARMACEUTICAL CO., LTD.

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�SS3-2Mass spectrometry-based omics approach reveals multifaceted roles of mitochondrial metabolism in the drug target, muscle differentiation, and maintaining its own DNA.Daiki Setoyama1）,�Dongchon�Kang1,2）

１）Clinical Laboratories, Kyushu University Hospital, 2）Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University

Mitochondrial metabolism has recently been described to play important roles not only in the development and progression of various diseases such as cancer, diabetes, cardiovascular disorders and neurodegenerative diseases but also in the process of stem cell/tissue differentiation and functional activation of immune systems. The remarkable progress in this field is largely due to the technological advance in systematically observing (mitochondrial) metabolic activities, such as flux analyzer and omics technology, the latter of which is to capture the whole picture of metabolic activity from comprehensively quantitative observation of the metabolites (metabolomics) and proteins (proteomics), respectively. We have continuously been developing a unique methodology based on our mass spectrometry platform in combination with metabolomics and proteomics to explore a novel aspect of mitochondrial metabolism. Herein, we first provide an overview of the technological advancement in this field and present our current research in terms of (1) identification of drug-target in mitochondria, employing the first-line drug for type 2 diabetes, metformin, (2) characterization of a mitochondrial metabolite alpha-ketoglutarate in myocardial and skeletal muscle differentiation in mouse C2C12 myoblast and P19.CL6 embryonal carcinoma cells, and (3) characterization of a novel link between the mitochondrial DNA replication and energy metabolism in human cells.

Sponsored Symposium 3: Mitochondria and Cardiac Metabolism Room 1 （Noh Theatre） September 22　15:10-16:40　Chairperson：Tetsuji�Miura（ Sapporo Medical University School of Medicine）� Tomomi�Ide（ Faculty of Medical Sciences, Kyushu University）SS3-1Mitochondrial fusion and fission proteins as targets for cardioprotectionDerek Hausenloy1）

１）Duke-National University Singapore

Mitochondrial health is critically dependent on the ability of mitochondria to undergo changes in mitochondrial morphology, a process which is regulated by mitochondrial shaping proteins. Mitochondria undergo fission to generate fragmented discrete organelles, a process which is mediated by the mitochondrial fission proteins (Drp1, hFIS1, Mff and MiD49/51), and is required for cell division, and to remove damaged mitochondria by mitophagy. Mitochondria undergo fusion to form elongated interconnected networks, a process which is orchestrated by the mitochondrial fusion proteins (Mfn1, Mfn2 and OPA1), and which enables the replenishment of damaged mitochondrial DNA. Emerging data supports a role for the mitochondrial shaping proteins in cardiac health and disease. Interestingly, in the adult heart, it appears that the pleiotropic effects of the mitochondrial fusion proteins, Mfn2 (endoplasmic reticulum-tethering, mitophagy) and OPA1 (cristae remodeling, regulation of apoptosis, and energy production) may play more important roles than their pro-fusion effects. In this talk, we provide an overview of the mitochondrial fusion and fission proteins in the adult heart, and highlight their roles as novel therapeutic targets for treating cardiac disease.


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�SS3-3Essential components to induce PINK1-Parkin mediated mitophagyAtsushi Hoshino1）

１）Kyoto Prefectural University of Medicine

Mitophagy is fundamental mechanism to eliminate dysfunctional mitochondria and maintain mitochondrial homeostasis. PINK1-Parkin mediated mitophagy is one of the programmed selective degradation systems for harmful mitochondria. Many gene mutations in this process are reported to be involved in wide range of diseases, indicating the importance of this mitochondrial quality control system. To understand the whole process of mitophagy, we performed CRISPR library screen with four different assay system and two mitochondrial stressors. This multidimensional screen identifies the majority of proteins reported to regulate mitophagy, reinforces the importance of ESCRT and HOPS machinery for autophagic degradation, and also discovers unknown essential components for PINK1/Parkin mediated mitophagy. As a receptor for LC3, Tax1bp1 dominantly recruits autophagosome in myoblast instead of optineurin and NDP52 that are primary receptors in HeLa cells. We also identify the TIM22 complex and the adenine nucleotide transporter (ANT) are fundamental mitochondrial components for PINK1 accumulation. Tim22 complex works as a critical chaperone for distribution of PINK1 to mitochondrial outer membrane, thereby forming complex with TOM import machinery, in which Tom5 and Tom7 contribute to PINK1 stabilization by its autophosphorylation. Upon mitochondrial bioenergetic collapse, closure of TIM23 is the initial step of PINK1 accumulation. ANT has protein-protein interaction with TIM23 and contributes to the inhibition of PINK1 import through TIM23, which is independent of its nucleotide translocase catalytic activity. Together, these data reveal the detailed mechanism underlying PINK1 accumulation to induce mitophagy.

�SS3-4The critical roles of coagulation factors in inducing brown adipose tissue dysfunction in obesity.Yuka Hayashi1),�Ippei�Shimizu2),�Yohko�Yoshida2),�Ryutaro�Ikegami1),�Goro�Katsuumi1),�Masayoshi�Suda2),�Tohru�Minamino1)

1）Department of CardioVascular Biology and Medicine Niigata University Graduate School of Medical and Dental Sciences, 2）Division of Molecular Aging and Cell Biology Niigata University Graduate School of Medical and Dental SciencesObese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model.Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improve Glucose intolerance and thermogenic response caused by metabolic stress. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. The inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in BAT in promoting dysfunction of this organ, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes.

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Sponsored Symposium 4Interorgan Communication Network

Abstract

Sponsored by MSD K.K.


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Sponsored Symposium 4: Interorgan Communication Network Room 2 （Conference Room 1・2） September 22　15:10-16:40　Chairperson：Thomas�N.�Sato（ Advanced Telecommunications Research Institute International）� Ichiro�Manabe（ Graduate School of Medicine, Chiba University）SS4-1Senescence, the endothelium and a vascular protective geneJennifer R Gamble1）,�Mathew�Vadas1）

１）Centenary Institute, Sydney

One of the major roles of endothelial cells is to maintain the anti-inflammatory, impermeable barrier of blood vessels. The ageing process is epidemiologically and causally linked to ongoing inflammation suggesting a dysfunction of aged endothelium. Senescent cells have been considered pro-inflammatory. However, we have identified a novel senescent phenotype in endothelial cells, an anti-inflammatory senescent cell as defined by their resistance to inflammatory stimuli. These anti-inflammatory senescent endothelial cells, which we have designed E2 cells, do not express adhesion molecules, nor secrete inflammatory cytokines, they fail to support leucocyte adhesion and they form an impermeable barrier. The mechanism of induction of the anti-inflammatory senescent endothelium is mediated through up-regulation of caveolae and suppression of NFkB. The anti-inflammatory senescent phenotype is induced by 3 of the major stresses of age: hypoxia, disturbed flow and oxidative stress as well as by overexpression of the gene, ARHGAP18 (SENEX). ARHGAP18 is highly expressed in both endothelial cells and smooth muscle cells. ARHGAP18 deficient mice show reduced senescence of the endothelium upon ageing. Further they show enhanced and accelerated atherosclerosis and increased susceptibility to thoracic aortic aneurysms, with heightened inflammation. Thus, we would suggest that ARHGAP18 is essential to mediate the anti-inflammatory phenotype of endothelial cells and smooth muscle cells and can be considered a vascular protective gene.

　SS4-2Organ specific gene regulation by distinct vascular mechanisms during development.Thomas N Sato1,2,3,4）,�Norio�Takada1,2）

１）The Thomas N Sato BioBEC-X Laboratories, Advanced Telecommunications Research Institute International (ATR), Kyoto, Japan, 2）ERATO Sato Live Bio-Forecasting Project, Japan Science and Technology Agency (JST), Kyoto, Japan, 3）Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA, 4）Centenary Institute, Sydney, Australia

Vascular system is essential for the development, function, and maintenance of organs by providing oxygen, nutrients and growth factors through the circulation. However, our recent transcriptional analysis indicated that the expression of sensory organ specific genes including olfactory marker protein a (ompa) is positively regulated by a local interaction between the organ and blood vessels in circulation-independent manner in developing zebrafish. This indication promoted us to hypothesize the existence of enhancer regulatory elements that are directly activated by blood vessels. To test this hypothesis, we performed a series of sequence bashing from 5’-upstream sequence to 3’-downstream using ompa genomic sequence and tested the reporter activity in developing zebrafish olfactory epithelium, where ompa is specifically expressed. Those experiments indicated that a combination of 1kb upstream sequence from a start codon and 2 kb sequence covering first intron is enough to drive the reporter expression in olfactory epithelium of wild-type embryos in endothelial cells-dependent manner, similar to the endogenous ompa expression. This observation suggests that ompa locus contains the regulatory sequences activated by blood vessels. Further characterization of such regulatory sequences is expected to better understand the molecular mechanisms of organ-vascular interaction and a role of vasculature in organ development.

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　SS4-3Immunometabolism in macrophage as a novel therapeutic target of metabolic syndromeYumiko Oishi1）

１）Nippon Medical School

Growing evidence has suggested that chronic inflammation is important for the pathogenesis of numerous diseases, including metabolic disorders and atherosclerosis. Among a variety of immune cells, macrophages play pivotal roles in the initiation and progression of those non-communicable diseases. Recently we found that macrophages switch their cellular metabolism and functional phenotype throughout the course of inflammatory response. In response to inflammatory activation via Toll-like receptor (TLR4), macrophages rapidly activate glycolysis, increase inflammatory cytokine expression, acquire M1-like, pro-inflammatory phenotype. By contrast, macrophages increase unsaturated, anti-inflammatory fatty acid synthesis to show M2-like, anti-inflammatory phenotype in the late inflammatory response at 24-48 hours following TLR4 activation. This late program of anti-inflammatory fatty acid biosynthesis is dependent on SREBP1 and results in the uncoupling of NFκB binding from gene activation. Consistent with this, anti-inflammatory omega-3 fatty acids are decreased in SREBP1-/- macrophages, and systemic inflammation was prolonged in SREBP1-/- mice. These findings suggest the functional switch from M1-like to M2-like, and the metabolic switch from glycolysis to lipid metabolism are tightly linked and coordinately regulated during inflammatory response, and these temporal regulatory programs are important for proper inflammatory activation and resolution (Oishi et al. Cell Metab 25:412, 2017). Interestingly, fatty acid desaturation was dysregulated in macrophages from aged animal, suggesting the metabolic dysregulation within macrophages may interfere with the resolution of inflammation associated with aging. Collectively, macrophages have endogenous, temporal programs to switch their function by linking inflammatory signals, and cellular metabolism. Macrophage's coordinated program would provide novel therapeutic targets for metabolic syndrome and atherosclerosis.

　SS4-4Specific Neural Stimulations Modulate the Formation of Immune Cell Gateways into the CNSDaisuke Kamimura1）,�Masaaki�Murakami1）

１）Molecular Psychoimmunology, Institute for Genetic Medicine, Hokkaido University

The blood-brain barrier (BBB) limits the exchanges of substances and the migration of immune cells to the central nervous system (CNS). Despite the BBB, immune cells can enter the CNS even at the steady state. In CNS inflammatory diseases such as multiple sclerosis (MS), the BBB is damaged and immune cell invasion occurs at a much greater degree. We found a mechanism operated by a specific local neuroimmune interaction that explains how autoreactive CD4+ T cells transverse the BBB to invade the CNS, which is now known as the gateway reflex. The gateway reflex is induced by the activation of specific neural pathways by various stimulations including gravity, electric, pain, and chronic stress etc, which enhances chemokine expressions at specific vessels and establishes specific gateways for autoreactive CD4+ T cells toward the CNS. Mechanistic analysis showed that neurotransmitters such as norepinephrine or ATP and cytokines at the specific vessels induce a simultaneous activation of NFkB and STAT3 followed by enhanced expression of various NFkB targets including chemokines to establish the gateway of immune cells in the BBB. While these gateways are localized at the vessels of the fifth lumbar spinal cord at the steady state due to stimulation by gravity, the location can be changed with the type of sensory stimulations. For example, pain induces an immune cell gateway at the ventral vessels of spinal cord, and electric stimulation creates gateways depending on where to stimulate. Recently, we unexpectedly found that chronic stress-mediated neural activation changes the location of immune cell gateway to from the fifth lumbar cord to the specific blood vessels of the brain, followed by a fatal dysregulation in the gastrointestine and heart. I will discuss the discovery and recent advances about the gateway reflex.


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Genetics / Epigenetics

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Sponsored Symposium 5: Genetics/Epigenetics Room 1 （Noh Theatre） September 23　9:00-10:30　Chairperson：Seiji�Takashima（ Graduate School of Medicine, Osaka University）� Koh�Ono（ Graduate School of Medicine, Kyoto University）SS5-1Developing new Treatments for CardiomyopathyJianming Jiang1）

１）National University of Singapore

Pathogenic variants in sarcomere genes cause the largest portion of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Dominant toxic gain-of-function or haploinsufficiency are disease mechanisms. Apart from typical treatments for broader cardiovascular disease, limited options are available for cardiomyopathy patients. Novel treatment development for cardiomyopathy is also limited due to lack of disease animal models and potential therapeutic targets. It is unclear whether targeting causal genes or downstream pathways serves as a therapeutic strategy for HCM and DCM. Here, I will use myosin heavy chain gene (MHC) related HCM and Titin gene (TTN) related DCM as examples to explore potential novel treatments for HCM and DCM. Our findings demonstrate that cardiomyopathy can be treated by modulation of causal genes or downstream signaling pathways.

　SS5-2Genetic analysis of more than 100K Japanese subjects including cardiovascular disordersYoichiro Kamatani1）

１）Center for Genomic Medicine, Kyoto University Graduate School of Medicine

Most of the common disorders including cardiovascular diseases are not determined by single gene mutation but affected by multiple genetic and environmental factors, thus they are called as complex diseases in a context of genetics. Although the initial comprehensive genetic study for such disease was reported by RIKEN using 94 Japanese myocardial infarction patients, most of the following large scale studies were performed in European population. It is important to establish genetic results in each ethnic sample to elucidate population specific genetic predisposition and for future precision medicine since a subject of different geographical origin has different genetic background. In this session I will explain achievements of BioBank Japan (BBJ), which is the largest genomic resource of non-European subjects in the world. We obtained genotypes at more than six million variant for 200,000 Japanese individuals in BBJ, and performed series of genome-wide association studies (GWAS). We identified ～ 200 susceptibility loci for body mass index (BMI) and a total number of more than 1400 loci for 58 laboratory tests or image test measures. We also participated in the international collaborative studies and found 97 susceptibility loci fo atrial fibrillation, and 32 loci for stroke. These large number of associations yielded us to reveal biological implications of them by using statistical approach, including the possibility of the involvement of CD19+ cells in the regulation of body weight. Finally, I will discuss the roles of genetic information in the clinical care of complex diseases.


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　SS5-4Single-nucleus RNA-seq analysis reveals pathogenic transcriptional programs in human heart failure.Kanna Fujita1),� Seitaro�Nomura1,2),� Takanori� Fujita2),�Masahiro� Satoh2),� Toshiyuki�Ko1,2),�Masamichi� Ito1),�Takashige�Tobita2),� Shintaro�Yamada1,2),�Hirotaka� Ieki1,2),�Xiaohui�Tian1,2),�Masaru�Hatano1),�Hiroyuki�Aburatani2),�Issei�Komuro1)

1）The University of Tokyo, Graduate School of Medicine, Cardiovascular Medicine, 2）Research Center for Advanced Science and Technology, The University of Tokyo

Single-cell transcriptome analysis is a powerful strategy uncovering heterogeneities of cells and their responses during developmental, physiological, and pathological processes. We have used single-cardiomyocyte RNA-seq analysis of the mouse heart after pressure overload, to reveal stress response heterogeneity, reconstruct the trajectory of cardiomyocyte remodeling, and show the transcriptional dynamics of the cardiomyocyte gene programs. However, it is difficult to simultaneously analyze transcriptomes of cardiomyocytes and non-cardiomyocytes in the heart because of the significant differences in cell size and RNA content among them.Here, we established a single-nucleus RNA-seq analysis pipeline to dissect cell-type composition and transcriptomic characterization in frozen heart tissues. Single-nucleus analysis of the frozen mouse heart classified cardiac cells at the molecular level, identified the uncharacterized marker genes, and revealed their heterogeneous expression patterns. By comparing single-nucleus RNA-seq profiles with single-cell RNA-seq profiles, we found a significant difference of mRNA localization between the cytosol and nucleus. mRNA of nuclear mitochondrial genes is preferentially localized in the cytosol, whereas that of extracellular matrix genes is in the nucleus. We also conducted single-nucleus RNA-seq analysis of the heart from patients with heart failure. We integrated single-cell gene expression profiles with the phenotypic characteristics such as treatment response and clinical prognosis, to reveal the molecular characteristics useful for cardiovascular precision medicine.

　SS5-3Practice and Application of Whole Exome Sequencing in Hereditary Heart DiseaseYoshihiro Asano1）

１）Osaka University Graduate School of Medicine

Next-generation sequence (NGS) analysis technology has made remarkable progress. It is necessary to establish a clinical sequence that extracts data necessary for diagnosis and treatment in the clinical setting. Meanwhile, from in the basic research field, establishing a platform for genomic medicine for rare diseases is an urgent issue, which can be overcome by identification of causative gene, elucidation of their functions, and development of molecular targeted drugs. NGS analysis technology shed light on solving the problem not only of cancer medicine, but also of medical treatment for rare intractable diseases. Here we introduce a practical example of genome medical development from basic study fields. We investigated a family with autosomal dominant bradyarrhythmias and conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating the transgenic animal model to confirm human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to up-regulate heart rate in bradyarrhythmias using the animal model. We conducted target resequencing in a genome cohort to find mutations in these genes, and have successfully identified additional rare mutations which are absent in any variation database. This study suggested that our results strengthen the association between these mutations and disease. Significant advances in drug discovery and development are sometimes based on identification of disease causing gene mutations in rare diseases. It is necessary to construct genome registry from the start point of the development of rare disease therapy. We hope that a new insight from such a novel gene identification will lead to clinical development of new drugs.

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Sponsored Symposium 6Vascular Biology

Abstract

Sponsored by Takeda Pharmaceutical Company Limited.


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　SS6-2Impact of vascular endothelial stem cells on vascular integrityNobuyuki Takakura1）

１）Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University

Existence of tissue specific stem cells has been elucidated in many organ, such as hematopoietic, neural, gut, epidermal (including hair) stem cells and so on. In the cardiovascular system, although cardiac stem cell population was identified by using cell surface markers, vascular endothelial stem cells (VESCs) have not been identified by their cell surface markers. We previously reported that endothelial cells (ECs) with highly proliferative potential was fractioned in side population (SP) cells which express ABC transporter abundantly. SP cells indeed differentiate into main population ECs and contribute to ECs for long term in hindlimb ischemia model; however, we could not show the hierarchy of ECs from stem cell population into terminally differentiated ECs through progenitor population. Here, we show that VESCs can be marked by CD157 and CD200 on cell surface. CD157 positive CD200 positive ECs generate higher amount of ECs compared to CD157 negative CD200 positive ECs and they differentiate into terminally differentiated CD157 negative CD200 negative ECs through CD157 negative CD200 positive progenitor like ECs having lower proliferating ability compared with CD157 positive CD200 positive ECs. Single CD157 positive CD200 positive EC could regenerate liver vascular unit for long-term. CD157 positive CD200 positive ECs continuously contributed to ECs in liver and rescued the bleeding phenotype of hemophilia A model mice by producing coagulation factor VIII. Therefore, we concluded that VESCs do exist in the pre-existing blood vessels and keep integrity in the vascular system. In this symposium, we will discuss about development and ageing of VESVs and involvement of VESCs in the vascular diseases.

Sponsored Symposium 6: Vascular Biology Room 2 （Conference Room 1・2） September 23　9:00-10:30　Chairperson：Toyoaki�Murohara（ Nagoya University Graduate School of Medicine）� Yoshiaki�Kubota（ Keio University School of Medicine）SS6-1TIE receptor mediated signaling in lymphatic and blood vascular formation and maintenanceYulong He1）

１）Soochow University

Tie receptors are important regulators in the establishment of lymphatic and blood vascular systems in mammals. Using conditional knockout mouse models targeting Tie1 and Tie2, we showed that Tie1 was crucial in the process of primitive lymphatic network formation as well as lymphatic remodeling to form collecting vessels and valves. In contrast, Tie2 signaling mediates blood vascular growth and maturation, and have a critical role in venogenesis. Specifically, vein formation was disrupted in mouse skin and mesentery when Tie2 signals were diminished by targeted deletion of Tie2 either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the formation of haemangioma-like vascular tufts in retina and venous tortuosity, via the Tie2-Akt pathway mediated stabilization of COUP-TFII. In spite of the essential role of Tie2 in blood vasuclar development, genetic deletion of Tie2 in neonate mice did not affect lymphatic vessel growth and maturation. However, we found that lymphatic vessels became dilated when Tie2 was deleted in endothelial cells at earlier embryonic stages. It remains to be investigated whether this is the primary effect of Tie2 in lymphatics or just secondary to tissue edema resulting from the impairment of blood vasculature.

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　SS6-4Role of thrombospondin-1 in mechanotransduction and development of thoracic aortic aneurysmYoshito Yamashiro1),�Hiromi�Yanagisawa1),�Bui�Quoc�Thang1),�Seungjae�Shin1),�Caroline�Antunes�Lino2),�Tomoyuki�Nakamura3),�Yuji�Hiramatsu1)

1）University of Tsukuba, 2）University of Sao Paulo, 3）Kansai Medical UniversityBackground: Abnormal mechanosensing of smooth muscle cells (SMCs) resulting from the defective elastin-contractile units has been suggested to drive the formation of thoracic aortic aneurysms (TAAs); however, the precise molecular mechanism has not been elucidated. Aim: The aim of this study was to identify the crucial mediator(s) involved in abnormal mechanosensing and propagation of biochemical signals during the aneurysm formation and to establish a basis for a novel therapeutic strategy. Methods and Results: We used a mouse model of postnatal ascending aortic aneurysms (Fbln4SMKO; termed SMKO), in which deletion of Fbln4 leads to disruption of the elastin-contractile units caused by a loss of elastic lamina-SMC connections. In this mouse, upregulation of early growth response-1 (Egr1) and angiotensin converting enzyme leads to activation of angiotensin II signaling. Here we showed that the matricellular protein, thrombospondin-1 (Thbs1), was highly upregulated in SMKO ascending aortas and in human TAAs. Thbs1 was induced by mechanical stretch and Ang II in SMCs, for which Egr1 was required, and reduction of Fbln4 sensitized the cells to these stimuli and led to higher expression of Egr1 and Thbs1. Deletion of Thbs1 in SMKO mice prevented the aneurysm formation in approximately 80% of SMKO; Thbs1-/- (termed DKO) animals and suppressed slingshot-1 and cofilin de-phosphorylation, leading to the formation of normal actin filaments. Furthermore, elastic lamina-SMC connections were restored in DKO aortas and mechanical testing showed that structural and material properties of DKO aortas were markedly improved.Conclusions: Thbs1 is a critical component of mechanotransduction as well as a modulator of elastic fiber organization. Maladaptive upregulation of Thbs1 results in disruption of elastin-contractile units and dysregulation of actin cytoskeletal remodeling, contributing to the development of ascending aortic aneurysms in vivo. Thbs1 may serve as a potential therapeutic target for treating TAAs.

　SS6-3The biological functions of thrombomodulin in endothelial cellsHua-Lin Wu1）,�Guey-Yueh�Shi1）

１）Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Thrombomodulin (TM) is a type I transmembrane glycoprotein that was first identified in the endothelial cells (ECs). It exhibits anticoagulant activity by forming a complex with thrombin to promote the activation of protein C. In the subsequent reports, TM was found in different cell types including keratinocytes, monocytes, smooth muscle cells and cardiomyocytes. TM was demonstrated to be involved in several biological functions. It functions as an adhesion molecule in conjunction with cadherin/occludin to stabilize cell-cell junctions in ECs and keratinocytes. We demonstrated that TM can regulate epithelial-mesenchymal transition. TM is a novel plasminogen (Plg) receptor that can regulate pericellular proteolysis and cell migration. Plg activation at the cell surface and the extent of its migration- and invasion-promoting effect are cellular TM expression dependent. Colocalization of TM, Plg and urokinase-type Plg activator at the leading edge of migrating ECs was demonstrated. In conclusion, TM has at least two opposite biological functions in ECs. It plays an important role in stabilizing the cell-cell junction in residing ECs. On the other hands, TM functions as a Plg receptor and promotes pericellular proteolysis in migrating ECs. The possible mechanisms involved in these two different distinct biological functions of TM will be discussed.


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Pulmonary Hypertension / Vasculitis

Abstract

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　SS7-2Inlerleukin-6 Blockade Therapy for Pulmonary Arterial Hypertension and Large Vessel VasculitisYoshikazu Nakaoka1）

１）Department of Vascular Physiology, National Cerebral and Cardiovascular Center Research Institute

Interleukin-6 (IL-6) is a multifunctional pro-inflammatory cytokine that is associated with the pathogenesis of various autoimmune diseases such as rheumatoid arthritis (RA). Humanized monoclonal anti-IL-6 receptor antibody tocilizumab (TCZ) is a biological agent which was approved for treatment of RA, juvenile idiopathic arthritis, and Castleman’s disease. However, the efficacy of TCZ for vascular diseases has not been elusive. Serum IL-6 level has been reported to correlate with the disease activity of pulmonary arterial hypertension (PAH) and Takayasu arteritis (TAK). We recently reported the possibilities of IL-6 blockade for treatment of PAH from basic study and for that of TAK from clinical study.For understanding the role of IL-6 in the pathogenesis of PAH, we examined the effect of IL-6 blockade on the hypoxia-induced pulmonary hyperntesion (HPH) in mice. IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated HPH. We identified IL-21 as a downstream target of IL-6 in HPH model. IL-21 receptor deletion in mice led to resistance to HPH almost similarly to IL-6 blockade. Consistently, significantly enhanced expression of IL-21 was detected in the lungs of idiopathic PAH patients who underwent lung transplantation. These findings suggest that IL-6/IL-21-signaling axis might be a novel potential target for treating PAH (PNAS 112(20): E2677-86, 2015).TAK is an autoimmune large vessel vasculitis which mainly affects the aorta and its major branches. Glucocorticoids (GCs), the first-line therapy for the treatment of TAK, are often associated with adverse effects when used long term or in high-dose, and patients frequently relapse during GC tapering. Other immunosuppressive agents may be used if relapse occurs while the patient is receiving GC; however, these agents did not demonstrate consistent clinical benefits or steroid-sparing effects. Thus, to examine the efficacy and safety of TCZ for TAK, we conducted a randomized, double-blind, placebo-controlled, phase 3 trial in Japan (the TAKT study). The results from the TAKT study suggest favor for TCZ over placebo for time to relapse of TAK without new safety concerns (Ann Rheum Dis. 77(3):348-354, 2018), leading to approval of TCZ for treatment of TAK in 2017 by the health authority in Japan.

Sponsored Symposium 7: Pulmonary Hypertension / Vasculitis Room 1 （Noh Theatre） September 23　14:00-15:30　Chairperson：Yoshikazu�Nakaoka（ National Cerebral and Cardiovascular Center Research Institute）� Noriaki�Emoto（ Kobe Pharmaceutical University）SS7-1VEGFR3 as a novel modulator of BMP signaling in pulmonary hypertensionSuk-Won Jin1）

１）Gwangju Institue of Science and Technology/Yale University

Bone Morphogenetic Protein (BMP) signaling has multiple roles in the development and function of the blood vessels. We have previously shown that BMP signaling provides venous specific pro-angiogenic cue in vertebrates. To better understand how responses toward BMP signaling in endothelial cells are modulated, we performed a combinatorial screen, and identified a number of genes which modulates the amplitude of BMP signaling in endothelial cells, including Vascular Endothelial Growth Factor Receptor3 (VEGFR3), which is the primary receptor for VEGF-C ligand. Here, we provide evidence that VEGFR3 functions as a key modifier of BMP signaling in the endothelium. We find that VEGFR3 is physical associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Moreover, conditional endothelial specific deletion of Vegfr3 in mice impaired BMP signaling responses and significantly worsened symptoms of hypoxia-induced pulmonary hypertension (PH). Consistent with these findings, pulmonary arterial endothelial cells isolated from human PH patients displayed substantially reduced VEGFR3 expression. Interestingly, reinstating VEGFR3 expression in these cells restored the sensitivity to BMP stimulation. Taken together, our findings suggest VEGFR3 may be a key determinant of PAH penetrance in humans with BMPR2 mutations.


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　SS7-3Cell-cell Contact Promotes Endothelial Integrity by BMPR2 Mediated Metabolic and Epigenetic ChangesKazuya Miyagawa1）

１）Kobe Pharmaceutical University

Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as pulmonary arterial hypertension. We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity. We found that in SMC and EC contact co-cultures, bone morphogenetic protein receptor 2 (BMPR2) is required by both cell types to produce collagen IV to releases Notch1 intracellular domain (N1ICD) to promote EC proliferation. Consistent with the findings in cultured SMC-EC contact co-cultures, in EC-SMC Bmpr2 double heterozygous mice, regeneration of carotid artery EC following injury is impaired, in keeping with reduced collagen IV production, decreased N1ICD and attenuated EC proliferation. EC regeneration is rescued in these mice by targeting N1ICD to EC. Deletion of EC-Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with loss of pre-capillary arteries and impaired EC proliferation. N1ICD maintains EC proliferative capacity by inducing the phosphofructokinase PFKFB3. ChIP-seq analyses showed that PFKFB3 is required for citrate-dependent histone acetylation at enhancer sites of genes regulated by the acetyl transferase p300, and primarily by N1ICD or the N1ICD target MYC, and necessary for EC proliferation and homeostasis. Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, to maintain monolayer integrity and vascular homeostasis in response to injury.

　SS7-4The single nucleotide polymorphism of MLX gene is involved in the development of Takayasu ArteritisYasuhiro Maejima1),�Natsuko�Tamura1),�Kenzo�Hirao1),�Mitsuaki�Isobe1)

1）Tokyo Medical and Dental University

Background: Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown etiology. We have identified that single nucleotide polymorphisms (SNPs) of MLX gene, which encodes MLX transcription factor, was significantly associated with clinical manifestations of TAK patients by genome-wide association study. The SNPs of MLX (rs665268) is a missense mutation of MLX that alters the Gln139 to Arg (Q139R). As Gln139 is located on the DNA binding site of MLX, we hypothesized that the mutation of Q139R on MLX, which alters the electric charge of the amino acid may enhance the formation of the MLX-DNA complex. Objective: We investigated whether an SNP of the MLX gene plays a critical role in the development of TA. Methods and Results: The SNP of MLX is significantly associated with the disease severity of TAK, including the morbidity of aortic regurgitation and the number of arterial lesions. Immunoprecipitation (IP) assays demonstrated that mutation of Q139R on MLX enhanced the heterodimer formation of MLX with MondoA, a binding partner of MLX that promotes thioredoxin-interacting protein (TXNIP) expression. Reporter gene assays and chromatin IP assays indicated that the mutation of Q139R on MLX is critical for the TXNIP promoter activity. Q139R mutation on MLX enhanced the protein level of both TXNIP and NLRP3 in human aortic smooth muscle cells. Treatment with IL-12 significantly enhanced the protein level of both TXNIP and caspase-1 in T-lymphocytes differentiated from human peripheral blood mononuclear cells of TAK patients with the risk allele for MLX compared to those from normal subjects. The activity of autophagy, a degradation system of inflammasome, is suppressed by Q139R mutation on MLX as well. Furthermore, MLX-Q139R mutant significantly enhanced the proliferation of macrophages and macrophage – endothelium interaction which was abolished by the treatment with SBI-477, an inhibitor of nuclear translocation of MondoA. Conclusion: Our current study suggest that SNP-mediated mutation of MLX on Gln139 would play a crucial role in the pathogenesis of TAK.

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Sponsored Symposium 8Arrhythmia

Abstract

Sponsored by Bayer Yakuhin, Ltd


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SS8-2Redox Physiology of TRP channelsYasuo Mori1）

１）Kyoto University Graduate School of Engineering

Ca2+-permeable cation channels encoded by the transient receptor potential (trp) gene superfamily are characterized by a wide variety of activation triggers that act from outside and inside the cell. Reactive species such as reactive oxygen species (ROS), reactive nitrogen species (RNS) and electrophiles are known to exert stress on organisms, but are also emerging as molecules that mediate cell signaling to regulate physiological responses. Understanding the physiological significance and activation mechanisms of TRP channel regulation by these reactive species has lead us to consider TRP channels as viable pharmacological targets; modulators of these channels may offer therapeutic options for previously untreatable diseases. Multiple TRP channels sense reactive species to induce diverse physiological and pathological responses, such as cell death, chemokine production, and pain transduction. Studies by numerous groups including us have revealed that sensitivity of TRP channels to reactive species either indirectly through second messengers or directly via oxidative modification of cysteine residues. In this session, I refer to these redox-sensitive TRP channels. Especially, I will focus on the TRPA1 channel and discuss its high sensitivity to molecular oxygen and related unique roles.

Sponsored Symposium 8: Arrhythmia Room 2 （Conference Room 1・2） September 23　14:00-15:30　Chairperson：Tetsushi�Furukawa（ Medical Research Institute, Tokyo Medical and Dental University）� Katsushige�Ono（ Faculty of Medicine, Oita University）SS8-1Cardiovascularpathophysiology of fatty acid binding proteinsWei Wang1）

１）Hebei Medical University

Background: Heart-type fatty acid-binding protein (FABP3) is abundantly expressed in cardiomyocytes and it is believed to play important roles in intracellular lipid trafficking. Recent studies revealed an unexpected role of FABP3 in suppressing calcium transients of cardiomyocytes. However, the mechanism is still elusive. Objective: The current study is aimed to determine the roles of FABP3 in regulating Sarcoplasmic Reticulum (SR) calcium release in mouse ventricular myocytes. Results: Confocal calcium imaging experiments revealed that FABP3 reduces evoked calcium transient amplitude and SR calcium content significantly in mouse ventricular myocytes in a dose dependent manner. Incidence of calcium wave and calcium spark frequency is significantly increased in FABP3-treated WT cardiomyocytes, suggesting FABP3 promotes RyR2-mediated SR calcium leak. At greater concentrations, FABP3 reduces calcium removal rate of evoked calcium transients, most likely due to its ability of promoting interaction between PLB and SERCA. His-tagged recombinant FABP3 can be detected within intact cardiomyocytes in a dose dependent manner. FABP3 localizes with both RyR2 and SERCA. The N-terminal peptide 1-20 (P1-20) of FABP3 largely retains the activity of full length FABP3 on RyR2, but has little effect on SERCA activity. Conclusion: FABP3 reduces SR Ca content in cardiomyocytes by promoting RyR2-mediated SR calcium leak and reducing SERCA activity.

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　SS8-4Single Cell Analysis of CALM-related LQT iPS Cell model using membrane voltage imaging systemYuta Yamamoto1),�Takeru�Makiyama1),� Seiko�Ohno2),�Yoshinori�Yoshida3),�Minoru�Horie4),�Naomasa�Makita5),�Takeshi�Kimura1)

1）Kyoto University, 2）National Cerebral and Cardiovascular Center, 3）Center for iPS Cell Research and Application, 4）Shiga University of Medical Science, 5）Nagasaki University

Background: Voltage sensitive dye has proven to be good tool to record action potentials (APs) in human iPS cell derived cardiomyocytes (hiPSC-CMs). However, there were few reports of single cell electrophysiological analysis in hiPSC-CMs using voltage sensitive dye because the signal intensity was low in fast responding dye used to record APs. Calmodulin (CaM) is a ubiquitous Ca2+ sensor molecule. Recently, mutations in CALM genes were reported to exert arrhythmias including long-QT syndrome (CALM-LQT), but there was no efficient treatment for CALM-LQT.Objectives: The present study aimed to establish the high-throughput single cell electrophysiological analysis system using voltage sensitive dye to develop the novel treatment for CALM-LQT.Methods: The hiPSC clones were generated from two LQTS patients carrying a heterozygous CALM2-N98S and CALM2-D134H (N98S and D134-hiPSC), and differentiated into cardiomyocytes. As a control, hiPSCs were generated from a healthy individual were used in this study. After loading hiPSC-CMs with Fluovolt (voltage sensitive dye), APs of single cardiomyocytes were recorded using membrane voltage imaging system.Results: The AP durations at 90% repolarization (APD90) of N98S (n = 12; 850.4 ± 46.9 ms) and D134H-hiPSC-CMs (n = 5; 765.4 ± 68.8 ms) were significantly longer than that of control (n = 15; 478 ± 48.5). In addition, beating rates of N98S (36.7 ± 3.0 bpm) and D134H (45.1 ± 7.2 bpm) were smaller than that of control (69.1 ± 8.9 bpm). Because APD was affected by beating rate, we corrected APD90 (cAPD90) by Bazett’s formula. Even after correction, the cAPD90s of N98S (648.7 ± 29.5 ms) and D134H (638.4 ± 29.5 ms) were significant longer than control (436.3 ± 41.4 ms). Conclusion: We successfully recapitulate the phenotype of CALM-LQT in different two types of CALM2 mutations using single cell membrane voltage imaging system. This imaging system may be useful for drug screening.

　SS8-3Extracellular nucleotides: Novel players for the pathogenesis of atrial fibrillationTetsuo Sasano1）,�Masahiro�Yamazoe2）,�Kensuke�Ihara2）,�Kenzo�Hirao1）,�Tetsushi�Furukawa2）

１）Tokyo Medical and Dental University, 2）Medical Research Institute, Tokyo Medical and Dental University

It is known that inflammation in atrial tissue is one of the critical factors for the progression of atrial fibrillation (AF), and the intercellular communications among atrial myocyte and other types of cells play a pivotal role for atrial inflammation. Recent findings have indicated that several kinds of nucleotides (DNA, RNA, and ribonucleotides) are released into the extracellular space, and act for intercellular and inter-organ communication. We have been investigating the function of these “extracellular nucleotides” in relation to the pathophysiological process of AF, and pursuing their possibility as biomarkers for predicting AF.Pathological role of extracellular nucleotides in AF Adenosine triphosphate (ATP) is a strong chemoattractant to recruit macrophage. The mechanical stretch on atrial myocytes released extracellular ATP through pannexin channel, followed by recruitment of macrophage. In vivo study showed atrial inflammation was evoked by pressure overload, which was diminished by blocker for pannexin. MicroRNA (miR), a small non-coding RNA regulates the expression of target genes. We found increased expression of miR in atrial tissue increased vulnerability of AF in high-fat diet treated mouse model. Cell-free DNA (cfDNA), which is released from apoptotic and necrotic cells, has attracted attention in inflammatory disorders. We focused on the contribution of cfDNA in AF. Electrical stimulation of atrial myocytes induced the release of cfDNA. The application of cfDNA, especially cfDNA from mitochondria (mt-cfDNA) induced the production of inflammatory cytokines in macrophages. Thus cfDNA may contribute to the systemic inflammation accompanied by AF.Extracellular nucleotides as possible biomarkers for AF The finding that several extracellular nucleotides were stable in circulating blood motivated us to use them for biomarkers. We performed a comprehensive analysis of the expression of miR in human serum with or without AF, combined with a comprehensive analysis of atrial tissue in murine AF-related models. We found 4 novel miRs had a significant association with the presence of AF. Another trial to measure cfDNA in circulating blood revealed that AF patients had higher plasma level of cfDNA and cf-mtDNA. These findings indicate miR and cfDNA may work as possible biomarkers for predicting AF.


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Grant Session for Basic Research: Room 1 (Noh Theatre) September 23　10:30-11:45　Chairperson：Naoki�Mochizuki�（ National Cerebral and Cardiovascular Center, Research Institute）� Keiichi�Fukuda（ Keio University School of Medicine）GS-1Systematic comprehension of developmental origins of adult-onset cardiovascular diseasesYuichiro Arima1）,�Kenichi�Tsujita1）

１）Department of Cardiovascular Medicine

The concept of developmental origins of health and diseases is that perinatal environmental stresses raise risks of non-communicable diseases. One of the remarkable clinical histories of perinatal stresses is low birth weight. Past epidemiological surveys and a recent genome-wide association study confirmed low birth weight (<2,500g) is a significant risk factor for cardiovascular diseases. The global prevalence of low birth weight is about 16% and one-tenth newborns are born as low birth weight babies in Japan. These evidences show low birth weight and perinatal environmental stresses could not be overlooked for adult cardiovascular diseases. However, underlying mechanisms are poorly understood.We have developed murine low birth weight models using calorie restriction and found several phenotypes in newborn. In liver, low birth weight newborns represented neonatal fatty liver and insufficient ketone body production was accompanied. Rate-limiting enzyme of ketogenesis, HMG-CoA Synthase 2 (HMGCS2) was transcriptionally suppressed and newly generated HMGCS2 KO recapitulated insufficient ketone body production and neonatal severe hepatic steatosis.In this project, we will build robust murine models of perinatal environmental stress and tackle to elucidate the etiological association between perinatal stresses and adult-onset cardiovascular diseases. In addition to calorie restriction based low birth weight model, premature delivery and combined methods will be introduced. For analysis, we have developed cell-type specific assessment of DNA methylation. We also analyze adult phenotypes using cardiovascular disease models.Thorough these techniques, we would like to unveil why adult cardiovascular disease susceptive trait develops by perinatal stresses. Our project will bring precise comprehension of developmental origins of health and diseases and serve the novel approach for preemptive medical care.

�GS-2New Insight into the Molecular Drug Target of Cardio-renal SyndromeTakuya Kumazawa1）,� Hitoshi�Nakagawa1),� Yasuki�Nakada1),� Tomoya�Nakano1),� Satomi�Ishihara1),�Kenji�Onoue1),�Yoshihiko�Saito1)

１）Cardiovascular Medicine, Nara Medical University, Japan

Patients with chronic kidney disease (CKD) are highly prone to cardiovascular diseases, including heart failure, cardiac hypertrophy, and atherosclerosis, named as the cardiorenal syndrome (CRS). However, the molecular mechanism underlying CRS remains unknown. Our previous study indicated that soluble Flt-1 (sFlt-1), an endogenous antagonist of placental growth factor (PlGF), as a key molecule of CRS. Progression of renal disease decreases sFlt-1 production and its deficiency causes atherosclerosis progression. Herein, we identified a novel long noncoding RNA (lncX) associated with exacerbation of CRS. lncX was specifically reduced after TAC in hearts of sFlt-1 KO mice. lncX KD increased proliferation of mouse embryonic cardiac fibroblast through controlling MAPK signaling. lncX inhibited MAPK signaling by binding to MEK in the cytoplasm. In vivo KD of lncX caused heart failure by pressure-overload as it aggravated cardiomegaly and lung to body weight ratios after TAC. Echocardiography revealed that left ventricular (LV) wall thickening was significantly increased and LV ejection fraction was decreased; histological analysis showed that myocardial fibrosis was significantly increased in lncX KD compared to that in the control. We are now focusing on four projects below to evaluate therapeutic potential of lincX to treat patients with CRS.1. Rescue experiment of lncX2. Identification of lncX functional element3. Analysis of lncX expression4. Identification of lncX homologus gene in human


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　GS-3Development of in vivo four-dimensional [4D] metabolism imagingYoshinori Katsumata1）,�Genki�Ichihara1）,�Yuki�Sugiura1）,�Jin�Endo1）,�Keiichi�Fukuda1）,�Motoaki�Sano1）

１）Keio University School of MedicineMatrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS); also referred to as mass spectrometry imaging, has been developed as a sensitive technique for in vivo imaging of individual biomolecules. To accurately assess highly labile metabolites, rapid inactivation of intracellular metabolism is critical. Extraction of the heart following cervical dislocation takes at least several seconds, rendering the oxidative phosphorylation-dependent heart tissue hypoxic; such circumstances significantly alters metabolites. To capture snapshots of acute metabolic changes, such as acute ischemia, we utilized focused microwave treatment to fix metabolic flow in vivo in hearts of mice. The left ventricle was subdivided into short-axis serial slices and the metabolites were analyzed by capillary electrophoresis mass spectrometry and matrix-assisted laser desorption/ionization imaging mass spectrometry. These techniques allowed us to determine the fate of exogenously administered 13C6-glucose and 13C3-lactate. Our previous data revealed that the penumbra regions, which are adjacent to the ischemic core, exhibited the greatest adenine nucleotide energy charge and an adenosine overflow extending from the ischemic core, which can cause ischemic hyperemia. Imaging analysis of metabolic pathway flows revealed that the penumbra executes accelerated glucose oxidation, with remaining lactate utilization for tricarboxylic acid cycle for energy compensation, suggesting unexpected metabolic interplays of the penumbra with the ischemic core and normoxic regions.However, continuously analyzing local in vivo metabolic changes in the hyper-acute phase of myocardial injury has been difficult to date. Understanding the metabolic changes in the hyper-acute phase of myocardial injury contributes to prevention of cardiac remodeling. Therefore, development of metabolic temporal/spatial imaging (in vivo four-dimensional [4D] metabolism imaging) technology is critical for understanding these metabolic changes.We aimed to continuously grasp temporal changes in local cardiac metabolism using an in vivo micro-dialysis method in mice hearts 10 min after ligation of the left anterior descending artery. An additional goal was to establish in vivo 4D metabolism imaging technology by combining this micro-dialysis technique and quantitative metabolic flux imaging (spatial grasp of metabolism) technology using isotope elements (13C palmitate, 13C glucose, 13C lactate, 13C acetoacetic acid, 13C glutamate, and 2H2).

　GS-4Mitochondria-nucleus network for a novel therapy against heart failureAtsushi Hoshino1）

１）Department of Cardiovascular medicine, Kyoto Prefectural University of Medicine

Mitochondria play a crucial role in cardiac function through energy production and calcium and redox metabolism. Mitophagy is a programmed autophagic degradation of damaged mitochondria and coupled with mitochondrial biogenesis to regulate mitochondrial content. It is well known that mitochondrial homeostasis is disrupted qualitatively and quantitatively during heart failure progression, however, crosstalk between mitochondria and nucleus is not fully understood. In this study, we try to comprehensively understand molecular network connecting mitochondria and nucleus using in vitro forward genetic screen and integrated analysis of transcriptome and epigenome in mitophagy inducible mouse. When mitophagy is induced robustly in cultured cells, mitochondrial content is temporary decreased and quickly recovers to the basal level. With CRISPR library screen in the assay reflecting this phenomenon, essential gene and pathway for mitochondrial biogenesis in response to mitophagy would be identified. It is assumed there are both PGC-1a, a master regulator of mitochondrial biogenesis, dependent and independent pathway. Mitophagy inducible mouse was originally developed here. Synthetic adaptor protein connecting mitochondria and autophagosome is expressed by the Tet-on system to genetically induce mitophagy. Mitophagy-upregulated heart has activated mitochondria-nucleus network to achieve compensatory mitochondrial biogenesis. Transcriptome and epigenome analysis of mitophagy-activated heart would identify upregulated molecular pathway to achieve high turnover of mitochondria. Failing heart has both lower mitochondrial biogenesis and lower mitochondrial content. Comprehensive understanding of molecular network between mitochondria and nucleus would provide novel therapeutic strategies for heart failure through the correction of impaired mitochondrial biogenesis and homeostasis.

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　GS-5Myocardial Regeneration by Engineering Mitochondrial TurnoverWataru Kimura1）

１）RIKEN Center for Biosystems Dynamics Research

Heart failure is a costly and deadly disease affecting over 23 million patients worldwide. The pathophysiological basis of heart failure is the inability of the adult heart to regenerate damaged myocardium. In contrast, the neonatal mammalian heart is capable of substantial regeneration following injury through proliferation of pre-existing cardiomyocytes. However, this regenerative capacity is lost by postnatal day 7, which coincides with cardiomyocyte binucleation and cell cycle arrest. Our data indicated that postnatal metabolic switching from glycolysis to mitochondrial oxidative phosphorylation taking place in cardiomyocytes mediates cell cycle arrest in the neonatal heart. Importantly, we have also shown that long-term exposure of adult mice to hypoxia induced suppression of mitochondrial respiration and cardiomyocyte cell cycle re-entry. These results highlight the importance of mitochondrial metabolism in cardiomyocyte cell cycle regulation, leading us to hypothesize that mitochondrial turnover, which is mediated by mitochondrial biogenesis and mitophagic degradation, is a key regulator of cardiomyocyte cell cycle. In this proposed research, we will investigate the roles of mitochondrial turnover on cardiomyocyte cell cycle regulation using pharmacological and genetic approaches. Firstly (aim 1), we will determine if pharmacological and genetic inhibition of mitochondrial biogenesis and mitophagy enhances/prevents cardiomyocyte cell cycle arrest in the neonatal heart. Secondly (aim 2), we will utilize the same models for the inhibition of mitochondrial biogenesis and mitophagy to test whether mitochondrial turnover enhances/prevents hypoxia-induced cardiomyocyte cell cycle re-entry in the adult heart. Successful completion of these aims uncover mitochondrial regulators that also mediate and maintain cardiomyocyte cell cycle arrest in the adult heart, which is an important step towards re-activating regenerative potential of the adult mammalian heart.


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Luncheon Seminar 1: Room 2 (Conference Room 1・2) September 22　12:45-13:45Chairperson：Michihiro�Yoshimura�（ Division of Cardiology, Department of Internal Medicine, The Jikei

University School of Medicine）LS1-1Environmental or Metabolic Factors Regulating Mitochondrial Respiration.Norihiko TakedaDepartment of Cardiovascular Medicine. Graduate School of Medicine, The University of Tokyo

LS1-2Deep Learning-Based System for the Research of Pluripotent Stem Cell-derived cellsShinsuke YuasaDepartment of Cardiology, Keio University School of Medicine

Luncheon Seminar 2: Room 3 (Conference Room 3・4) September 22　12:45-13:45Chairperson：Yasushi�Sakata�（Osaka University）

LS2Takuya�KishiKyushu University


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Luncheon Seminar 3: Room 4 (Reception Hall 1) September 22　12:45-13:45Chairperson：Hiroyuki�Tsutsui�（Kyushu University）

LS3Onco-Cardiology: Exploring a New Frontier with Basic CardioVascular ResearchHiroshi AkazawaThe University of Tokyo

Cancer remains to be the leading cause of death in our country, but recent progress in cancer therapy has improved the long-term outcome for cancer patients. On the other hand, the patients suffering from cardiovascular diseases are increasing explosively in number because of the westernized lifestyles and progressive population aging. Under such circumstances, cardiovascular diseases become responsible for substantial morbidity and mortality, and it is increasingly of importance to manage the cardiovascular complications in cancer patients or survivors, which are related to both cancer itself and adverse effects of cancer therapies. Among the most concerning as cardiovascular complications of cancer therapies are cancer therapy-related cardiac dysfunction, coronary artery disease, venous thromboembolism, severe hypertension, lethal arrhythmias, peripheral vascular disease and stroke, and pulmonary hypertension, which occasionally lead to unwilling interruption of cancer therapy. Unfortunately, definitive strategies to predict, prevent, and treat these complications are currently lacking. To solve the unmet needs, interdisciplinary collaboration is required to integrate the many different perspectives of clinicians, basic researchers, regulatory scientists, co-medical workers, and even patients. Especially, basic cardiovascular research has a major role in defining the pathogenic mechanisms, and establishing diagnostic strategies with risk prediction and evidence-based therapeutic strategies for cardiovascular complications of cancer therapies.

Luncheon Seminar 4: Room 2 (Conference Room 1・2) September 23　12:00-13:00Chairperson：Issei�Komuro�（ Department of Cardiovascular Medicine, Graduate School of Medicine, The


LS4Shigeo HorinakaDepartment of Cardiology and Nephrology, Dokkyo Medical University

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Luncheon Seminar 5: Room 3 (Conference Room 3・4) September 23　12:00-13:00Chairperson：Tetsuji�Miura（Department of Cardiovascular, Renal and Metabolic Medicines）

LS5Understanding SGLT2 Inhibitors from the Perspective of Drug Development –The Clinical Possibil it ies of Pharmacological Characteristics and Mitochondrial Metabolism – Masashi�SuganumaSuganuma Clinic

SGLT2 is responsible for the reabsorption of glucose in the proximal renal tubules; SGLT2 inhibitors are the drugs that lower blood glucose levels through the inhibition of this glucose reabsorption and therefore increase the secretion of glucose in the urine. Currently, seven products are available in Japan, of which six are pharmaceutical preparations. The basic structure of all SGLT2 inhibitors contains a C-glycoside derivative, but they possess different side chains, which are thought to be responsible for a variety of pharmacological and pharmacokinetics profiles.In this presentation, I would like to discuss the expectations for and the possibilities of the use of SGLT2 inhibitors in clinical practice, from the perspective of my extensive involvement in drug development.


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AS-2Increased Gαo expression underlies cardiac dysfunction accompanied with abnormal Ca2+ handlingHideaki Inazumi1),�Koichiro�Kuwahara2),�Yoshihiro�Kuwabara3),�Yasuaki�Nakagawa1),�Hideyuki�Kinoshita1),�Kenji�Moriuchi1),�Hiromu�Yanagisawa1),�Toshio�Nishikimi1,4),�Miku�Oya2),�Mitsuhiko�Yamada5),�Toshihide�Kashihara5),�Nagomi�Kurebayashi6),�Kazuwa�Nakao7),�Takeshi�Kimura1)

1）Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 2）Department of Cardiovascular Medicine, Shinsyu University School of Medicine, 3）Center for ACCessing Early Promising Treatment, Kyoto University Hospital, 4）Wakakusa-Tatsuma Rehabilitation Hospital, 5）Department of Molecular Pharmacology, Shinsyu University School of Medicine, 6）Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of medicine, 7）Medical Inovation Center, Kyoto University Graduate School of MedicineBackground: We previously demonstrated that a transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability by repressing fetal cardiac gene expression by using transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg), which exhibit systolic dysfunction with cardiac dilation and premature death due to lethal arrhythmias. However, precise molecular mechanisms, by which NRSF maintains normal cardiac systolic function, remains unknown. Methods and Results: We have generated cardiac-specific NRSF knockout mice (NRSFcKO) by using mice with cardiac-specific cre expression driven by αMHC promoter, and confirmed that cardiac phenotypes of NRSFcKO are similar to those of dnNRSF-Tg. cDNA microarray analysis revealed that cardiac gene expression of GNAO1 that encodes Gαo, a member of inhibitory G protein Gαi family, is commonly increased in both dnNRSF-Tg and NRSFcKO ventricles. We also identified gene expression of GNAO1 is increased in hypertrophied murine hearts induced by transverse aortic constriction. Furthermore, we confirmed that NRSF binding sequences are located in the second intron of Gαo gene, and found that GNAO1 gene is a direct target of NRSF by using ChIP-seq data, reporter assay and electrophoretic mobility shift assay. In dnNRSF-Tg, pharmacological inhibition of Gαo with pertussis toxin improved systolic dysfunction and knockdown (KD) of GNAO1 by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates. We found that KD of GNAO1 ameliorates abnormalities in Ca2+ handling, including increased current density in surface sarcolemmal L-type Ca2+ channel, reduced content of sarcoplasmic reticulum Ca2+, lowered peak of Ca2+ transient, and accelerated abnormal automaticity, which are observed in ventricular myocytes of dnNRSF-Tg. Moreover, KD of GNAO1 attenuated increased phosphorylation levels of CAMK2 in dnNRSF-Tg, which presumably underlies the improvement in Ca2+ handling. Conclusions: We identified that increased expression of Gαo, induced by attenuation of NRSF-mediated repression, plays a crucial role in the progression of cardiac dysfunction and lethal arrhythmias by evoking Ca2+ handling abnormality, providing a potential therapeutic target for heart failure.

Award Session Room 1 （Noh Theatre） September 22　13:55-15:10　Chairperson：Tetsuo�Minamino（ Faculty of Medicine, Kagawa University）� Masaki�Ieda（Faculty of Medicine, University of Tsukuba）AS-1Cell-free DNA Released in Atrial Fibrillation Promotes Systemic Pro-Inflammatory Cytokine ExpressionMasahiro Yamazoe1),�Tetsuo�Sasano2),�Wakana�Nakamura2),�Kensuke�Ihara1),�Naomi�Takahashi2),�Kenzo�Hirao3),�Tetsushi�Furukawa1)

1）Department of Bio-informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2）Department of Biofunctional Informatics, Tokyo Medical and Dental University, 3）Department of Cardiovascular Medicine, Tokyo Medical and Dental UniversityIntroduction:Atrial fibrillation (AF) is often accompanied with systemic inflammation; however, the underlying mechanism remains unclear. Recently, “cell-free DNA (cfDNA)” which is released from cells and circulating in the blood, has been attracted attention in relation to inflammatory disorders. We aimed to evaluate the cfDNA in patients with AF and its impact on inflammation, especially focusing on the difference of cell-free nuclear (cf-nDNA) and mitochondrial DNA (cf-mtDNA).Methods:Peripheral blood was obtained from 39 AF patients and 36 controls. We extracted cfDNA from plasma, and absolute copy number of cf-nDNA and cf-mtDNA were calculated with quantitative PCR. In in vitro analysis, we extracted total-cfDNA from condition medium after rapid pacing to HL-1, murine atrial cardiomyocytes. Additionally, nuclear and mitochondrial DNA were separately extracted from mouse liver and fragmented by sonication to simulate cf-nDNA and cf-mtDNA. These cfDNAs were applied on J774.1, murine macrophage, followed by quantitative reverse transcription-PCR to evaluate the expression level of pro-inflammatory cytokines. Further, we evaluated whether this response is through Toll-like Receptor 9 (TLR9) recognizing CpG DNA, or not.Results:The AF group had higher cfDNA concentration than non-AF group (14.0±9.0 vs. 8.8±5.1 [ng/ml], p=0.007). The copy number of cf-mtDNA was higher in AF groups than non-AF groups (p<0.001), but that of cf-nDNA did not show significant difference. Administration of total-cfDNA on macrophages significantly promoted IL-1β and IL-6 expression levels, whereas those of TNFα and MCP-1 were not changed. Similar significant increase in IL-1β and IL-6 were observed by administration of cf-mtDNA, but not by cf-nDNA. In addition, TLR9 antagonists attenuated the IL-1β and IL-6 induction by cf-mtDNA.Conclusions:AF was associated with increased in cfDNA level, especially in cf-mtDNA. cf-mtDNA released from cardiomyocytes might be involved with sterile systemic inflammation accompanied with AF.


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AS-3Direct Reprogramming with Sendai Virus Vectors Improves Cardiac Function In Vitro and In VivoKazutaka Miyamoto1),�Fumiya�Tamura1),�Mari� Isomi1),�Naoto�Muraoka1),�Taketaro�Sadahiro1),�Keiichi�Fukuda1),�Masaki�Ieda2)

1）Keio University School of Medicine, 2）Tsukuba University School of MedicineDirect cardiac reprogramming holds great promise for regenerative medicine. We previously demonstrated that fibroblasts were directly reprogrammed into induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, the technique has been inefficient and the molecular mechanism of cardiac reprogramming remains unclear. Moreover, these genome-integrating vectors may cause insertional mutagenesis. So, these problems has blocked the clinical application of cardiac reprogramming strategy with retrovirus vectors. Here, we developed cytoplasmic type RNA vectors, Sendai viral vectors (SeV), which overexpress cardiac reprogramming factors without the risk of insertional mutagenesis, and these vectors could efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. Transduction of a polycistronic SeV-Gata4/Mef2c/Tbx5 (SeV-GMT) greatly promoted cardiac reprogramming, and induced spontaneously beating iCMs in the ~40% of the transduced fibroblasts, which was 100-fold more than that with the conventional retroviral-mediated cardiac reprogramming. Moreover, SeV-GMT shortened the duration to generate beating iCMs from 30 to 10 days, demonstrating rapid and robust cardiac induction with SeV-GMT. Functionally, we showed that SeV-GMT-iCMs consisted of three subtypes of functional cardiomyocytes, mainly atrial-type iCMs recording action potentials of beating iCMs by patch clamping. Mechanically, the robust and optimal transgene expression by SeV-GMT was the critical factor contributing to the efficient cardiac reprogramming, and we showed SeV-GMT strongly induced and upregulated endogenous Gata4, Mef2c, and Tbx5 expression and multiple cardiac genes over time for 4 weeks in MEFs.Next, in vivo cardiac reprogramming, we injected SeV-GMT into infarct region of mouse hearts after coronary ligation. Immunostaining revealed that SeV-GMT could induce cTnT+ and α-actinin+ iCMs more rapidly and efficiently than retroviruses. Injection of SeV-GMT greatly improved ejection fraction and reduced fibrosis after myocardial infarction. Consequently, this strategy raised survival rate. Thus, we develop a new, efficient and safe vector for cardiac reprogramming that may open an avenue for clinical applications.

AS-4Blockade of NKG2D / NKG2D Ligand Interaction Attenuated Post-infarct Myocardial InjuryKotaro Matsumoto1),�Masanori�Obana1),�Makiko�Maeda1),�Yasushi�Sakata3),�Hiroyuki�Nakayama1),�Yoshiki�Sawa2),�Yasushi�Fujio1)

1）Laboratory of Clinical Science and Biomedicine, Graduate School of Pharmaceutical Sciences, Osaka University, 2）Department of Cardiovascular Surgery, Graduate School of Medicine, Osaka University, 3）Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka UniversityBackground:The pathogenesis of heart failure (HF) is closely associated with immune reactions, though molecular mechanisms remain to be fully elucidated. Here, we focused on immunosurveillance system that induces apoptotic cell death in cytopathic cells through the interaction between Natural Killer Group 2 member D (NKG2D) on immune cells and NKG2D ligands (NKG2DLs) on cytopathic cells. The aim of this study is to clarify the pathophysiological significance of immunosurveillance system in HF. Methods and Results:Murine myocardial infarction (MI) was generated by the occlusion of left anterior descending artery (LAD). The expression of NKG2D and NKG2DLs, especially Retinoic acid early induced transcript-1ε(Rae-1ε), was concomitantly increased with the peak 3-7 days after MI (12.2±11.0, 24.7±8.6 vs non MI, respectively, n=5-9). Immunohistochemistry revealed that Rae-1 was expressed on the membranes of injured cardiomyocytes in the infarct and border area. In addition, Rae-1 expression was mediated through p53 because Rae-1 expression was suppressed by 54.7±16.9% in post-MI p53-/- mice and the overexpression of p53 increased Rae-1 expression in cultured cardiomyocytes. Flow cytometric analysis showed that NKG2D-expressing cells in post-MI myocardium were mainly γδT cells. The co-culture of Rae-1ε-expressing cardiomyocytes and γδT cells increased the frequency of apoptotic cells by 13.9±3.6 % in the cultured cardiomyocytes, compared with control. Importantly, intraperitoneal injection of the blocking antibody against Rae-1ε into post-MI mice exhibited cardioprotective effects with reduced apoptotic myocytes (9.1±2.9 vs 3.5±2.1 cells/mm2, n=5), suppressed cardiac fibrosis (43.1±10.0% vs 30.8±8.7%, n=8-12), and attenuated cardiac dysfunction (FS: 30.6±4.3% vs 41.9±2.0%, n=5-6). Finally, tamoxifen-inducible cardiomyocyte-specific Rae-1ε overexpressing mice were susceptible to post-infarct remodeling with the increase of cardiomyocyte apoptosis (11.2±1.5 cells/mm2 vs 27.9±10.8 cells/mm2, n=5) and severer cardiac dysfunction. Conclusion:Cytopathic cardiomyocytes were eliminated by immunosurveillance system in post-infarct cardiac remodeling. Blockade of NKG2D/NKG2DL interaction could be a promising therapeutic strategy against HF.

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　AS-5(Pro)renin receptor promotes development of sarcopenia via activation of Wnt- YAP signaling pathwayJin Endo1),�Naohiro�Yoshida2),�Atsuhiro�Ichihara2),�Keiichi�Fukuda1),�Motoaki�Sano1)

1）Keio University School of Medicine, 2）Tokyo Women’s Medical University

Developed countries are currently facing rapid growth of the aging population. To extend healthy life expectancy, prevention and treatment of age-related skeletal muscle atrophy, also known as sarcopenia, is crucial. However, the molecular mechanisms underlying sarcopenia remain unclear. The Wnt/beta-catenin signaling pathway promotes age-related phenotype and has been recently identified to be activated by (Pro)renin receptor ((P)RR). We revealed that (P)RR protein expression was increased in the atrophied skeletal muscles of aged mice and humans. To elucidate whether the increase in (P)RR expression affected the muscle mass and strength, we developed (P)RR-transgenic (Tg) mice by expression of mouse ATP6AP2/(P)RR under control of the CAG promoter. Consistent with our hypothesis, (P)RR-Tg mice died early and exhibited muscle atrophy with the histological features of sarcopenia including small circular fibers, central nucleus, and selective decrease in fast-twitch fibers. The expression of p16, p21 and gamma-H2AX, the representative markers of senescence, were significantly increased in skeletal muscles in (P)RR-Tg mouse. In (P)RR-Tg mice, Wnt/beta-catenin signaling was activated and the regenerative capacity of muscle progenitor cells after cardiotoxin injury was impaired due to cell fusion failure. And in vitro study showed that forced expression of (P)RR protein in C2C12 myoblast cells suppressed myotube formation via augmentation of the Wnt/beta-catenin signaling. Using Dickkopf-related protein 1 (DKK1), an inhibitor of beta-catenin pathway, we demonstrated that myogenic differentiation was disturbed by the activation of Wnt/beta-catenin signaling in (P)RR-expressing C2C12 myoblast cells. Also in in vivo, DKK1 and anti-(P)RR neutralizing antibody significantly attenuated sarcopenia in (P)RR Tg mice. Further, YAP/TAZ signaling which is known to be coordinately regulated by Wnt/beta-catenin signaling contributed to the development of (P)RR-induced sarcopenia. The present study demonstrated that (P)RR-Tg mouse was a novel sarcopenia model, and (P)RR-Wnt-YAP signaling pathway played a major role in the pathogenesis of sarcopenia. Blocking agents against (P)RR-Wnt-YAP signaling represents a promising therapeutic candidate for sarcopenia.


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Abstract

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P01-4Endothelial cell senescence exacerbates heart failure due to pressure-overloadDhite B Nugroho1,2),� Koji� Ikeda3),� Agian� J� Barinda3),�Pranindya�Rinastiti1),�Noriaki�Emoto1,3)

1）Department of Internal Medicine, Division of Cardiovascular Medicine, Graduate school of medicine, Kobe University, 2）Departement of Internal Medicine, Gadjah Mada University, 3）Department of Clinical Pharmacy, Kobe Pharmaceutical UniversityAdvanced age is a significant risk factor for cardiovascular diseases such as hypertension and cardiac hypertrophy. The vascular system forms an essential component of cardiac tissue, to provide routes for circulation and transportation of nutrients and oxygen throughout the cardiac muscle. In addition to its function in vascular biology such as vasodilation and neovessel formation, endothelial cell (EC) also provides many secreted angiocrine factors that are crucially involved in maintaining tissue homeostasis. Aging induces cellular senescence in various cells including EC. Senescent cells produce senescence-messaging secretome that has deleterious effects on the tissue microenvironment, referred to as the senescence-associated secretory phenotype (SASP). Because of the crucial roles of EC in tissue homeostasis, EC senescence is presumed to play significant roles in age-related cardiac dysfunction, however, whether and the mechanism by which EC senescence affects age-related cardiac dysfunction remains to be elucidated. In this study, we investigated the role of senescent ECs in cardiac hypertrophy and heart function by utilizing EC-specific progeroid mice that overexpress the dominant negative form of telomeric repeat binding factor 2 under the control of the VE-cadherin promoter (VEcad-TRF2DN-Tg). The serial echocardiographic analysis after transverse aortic constriction (TAC) revealed the exacerbated left ventricular dysfunction in VEcad-TRF2DN-Tg compared to that in wild-type mice . Morphometric and histological analysis 4 weeks after TAC showed increased heart weight and aggravated cardiac fibrosis in VEcad-TRF2DN-Tg mice. Moreover, we found that treatment with conditioned medium derived from senescent ECs, which was enriched with senescence-messaging secretome, enhanced cardiomyocyte hypertrophy in H9C2 cells in vitro. These findings demonstrate for the first time that senescent ECs play causative roles in age-related cardiac disorders through the SASP.

P01-1Nucleolus-mediated Cellular Senescence is involved in Heart FailureTakuya Kumazawa1), � Kazuho� Nishimura2), � Hitoshi�Nakagawa1),�Yasuki�Nakada1),�Tomoya�Nakano1),�Satomi�Ishihara1),�Kenji�Onoue1),�Yoshihiko�Saito1)

1）Cardiovascular Medicine, Nara Medical University, Japan, 2）Graduate School of Life and Environmental Sciences, University of Tsukuba, JapanIn response to various stresses, the tumor suppressor p53 initiates stress response programs and thus selects cell fates. Post-translational modifications and binding partners of p53 are considered central to various transcription programs that determine cell fate. However, upstream sensors of stress signal-transduction cascades remain unknown. In this study, we show that the nucleolus regulates post-translational modifications and binding partners of p53 by altering nucleolar RNA content, which eventually discriminates between apoptosis and cellular senescence. Apoptosis-inducing stresses reduced nucleolar RNA content by inhibiting rRNA transcription1. However, senescence-inducing stresses increased nucleolar RNA content by accelerating rRNA transcription, or by inhibiting rRNA processing2. In addition, experimental regulation of nucleolar RNA content discriminated between apoptosis and cellular senescence. Specifically, upon reduction of nucleolar RNA content, ribosomal protein L11 (rpL11) accumulated p53 and the nucleolar protein Myb-Binding Protein 1A (MYBBP1A) enhanced acetylation of p53. In contrast, elevated nucleolar RNA content led to accumulation of p53 via rpL11 and Arf. Taken together, fluctuations of nucleolar RNA content determine p53 activity by coordinating the release of nucleolar proteins, and eventually decide individual cell fates.Recently, the contribution of p53 to many undesirable aspects of aging and age-associated diseases, such as cardiovascular and metabolic disorders, has been recognized. It has been reported that aging is associated with p53-mediated cellular senescence and that slight constitutive activation of p53 is associated with premature aging in mice. p53 activation and p53 induced-cellular senescence have also been observed in aged blood vessels and failing hearts and have been implicated in atherosclerosis and heart failure. Therefore, we also examined the role of nucleolus mediated-cellular senescence in heart failure. We found that elevated nucleolar RNA content caused hypertrophic growth of cardiomyocytes. Hypertrophy of cardiomyocytes is associated with heart failure. Moreover, using coculture system, we also found that senescent cardiac fibroblast induced by increasing nucleolar RNA content promoted hypertrophy of cardiomyocytes. These results implied that nucleolus mediated-cellular senescence contribute to exacerbation of heart failure. Thus, modulation of the nucleolus may represent a novel approach for treatment of heart disease.

P01-2Amino ac id subst i tut ion of Werner gene is responsible for cardiac agingTakahiro Kamihara1),� Yasuko� K� Bando1),� Kazuyuki�Nishimura1),�Toyoaki�Murohara1)

1）Department of Cardiology, Nagoya University Graduate School of Medicine[Background/Introduction] Werner syndrome is one of the aging disorder caused by dysfunction of the DNA helicase-regulatory protein (WRN). Amino acid (AA) substitution of WRN at position 577 (WRN-K577M) has been reported to abolish the ATPase andhelicase activities and its mutant mice exhibits accelerated skin aging. Our aim is to elucidate whether WRN-K577M is responsible for cardiac aging in mice.[Methods] C57/BL6-based mutant mice harboring WRN-K577M were evaluated at 18 week-old and 84 week-old.[Results] At 18-week-old (18w-WRN-K577M), appearance of WRN-K577M was normal. However, cardiac aging markers (p53 and γH2AX) and apoptosis detected by TUNEL were augmented in 18w-WRN-K577M. 18w-WRN-K577M exhibited cardiomegaly and diastolic left-ventricular (LV) dysfunction, whereas their systolic LV function was preserved, with concomitant cardiac fibrosis and hypertrophy.Consistently, hypertrophy-associated signaling was elevated (mTOR in WRN-KD versus CON; 1.4 fold, AKT; 1.4 fold, ERK; 1.4 fold). DNA microarray analysis of 18w-WRN-K577M heart revealed that the 253 genes was upregulated compared to wild-type. Among them, 16 genes were increased > 4 fold higher than wild. KEGG ontology revealed that characteristics of these genes were as follows: hypertrophy (Myh7, Klkb11), fibrosis (Fgf21, Ctgf), and inflammatory molecules (Ap1s3, Pla2g2e, Has1, MMP9). In contrast, at 84-week-old, WRN-K577M exhibited significant hair loss, retarded locomotive behavior, systolic LV dysfunction (LVFS; 26.2±2%) and decline in LV wall thickness with consistent decrease in cardiomyocyte size and increase in cardiac fibrosis and apoptosis.[Conclusion(s)] We report the impact of WRN-K577M on chronical cardiac aging. The WRN-K577M at earlier age is responsible for diastolic LV dysfunction related to cardiac hypertrophy and fibrosis via enhanced gene alteration related to cardiac remodeling and inflammation. The WRN-K577M mutation is responsible for not only phenotype of systemic progeria but also for transition from the diastolic to systolic LV dysfunction observed in progeria.

P01-3Endothelial-SASP induces premature senescence in adipocytes and disrupts metabolic homeostasisAgian Jeffilano Barinda1,2),� Koji� Ikeda1),� Dhite� Bayu�Nugroho1,2),�Donytra�Arby�Wardhana1,2),�Keiko�Yagi1),�Ken-ichi�Hirata2),�Noriaki�Emoto1,2)

1）Clinical Pharmaceutical Laboratory, Kobe Pharmaceutical University, 2）Cardiovascular Medicine Division, Kobe University

Aging causes dysfunction of organs including adipose tissues and blood vessels, which is associated with age-related diseases. During ageing, senescent cells secrete many factors that impair surrounding healthy cells, termed as senescence associated secretory phenotype (SASP). Adipose tissues possess well-developed vascular networks; however, potential roles of senescent endothelial cell (EC) in age-related adipose tissue dysfunction remain unknown. Here, we revealed a role of senescent EC in adipocyte functions and systemic metabolic homeostasis through senescence-associated secretory phenotype (SASP). Treatment of 3T3-L1 adipocytes with a conditioned medium derived from senescent EC induced premature senescence in adipocytes due to enhanced oxidative stress. These premature senescent adipocytes exhibited a reduction in IRS-1 expression, leading to impaired insulin signaling. To investigate a role of endothelial SASP in vivo, we generated endothelial progeroid mice in which ECs are specifically senescent through overexpressing dominant-negative form of telomeric repeat binding factor-2 (TRF2DN) under the control of the Tie2 promoter (Tie2-TRF2DN-Tg). Tie2-TRF2DN-Tg mice showed premature senescence of white adipocytes in association with enhanced oxidative DNA damage, and exhibited impaired insulin sensitivity as early as 20 weeks old even under normal dietary conditions. Shared circulation with endothelial progeroid mice by parabiosis sufficiently transmitted the adipocyte premature senescence and metabolic dysfunction into wild-type recipient mice. Our data provide direct evidence that EC senescence disrupts metabolic function in adipocytes through the SASP, which could play a causative role in age-related metabolic disorders.


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P01-5Repetitive Postprandial Combined Metabolic Spikes Induce Premature Aging of Bone MarrowKeita Horitani1),�Masayoshi� Iwasaki1),�Hiroshi�Kishimoto1),�Kensaku�Wada1),�Ichiro�Shiojima1)

1）Department of Medicine II Kansai Medical UniversityGlucose and fatty acid metabolism strongly affect stem cell behavior and patients with type 2 diabetes (T2DM) exhibit dysregulated glucose and fatty acid metabolism. In this study, we aimed to explore how the variability of blood glucose and triglyceride level affect homeostasis of endothelial progenitor cells (EPC) in bone marrow (BM). First, to find the factors that strongly affect EPC homeostasis, we carried out meal test in subjects with HbA1c level of 5.6 to 7.6%, and examined the correlation between metabolic indices and the number of circulating EPC. Multiple regression analysis revealed that post meal test glucose level at 60min is a determinant of the number of circulating EPC. Of note, the presence of post meal test hypertriglyceridemia further improved the correlation. We therefore examined the effect of repetitive glucose/triglyceride spikes on BM in experimental studies by injecting glucose (G) and/or lipid emulsion (L) in C57BL/6 mice for 7 days. Compared to saline-injected controls, BM in mice injected with G+L exhibited characteristic features of premature aging (i.e., increased lin-sca-1+c-kit+ (LSK) cell fraction and myeloid-biased differentiation). Furthermore, repetitive G+L injection attenuated BM stem cell quiescence and function. We found that repetitive G+L injection also downregulated Sirtuin 1 in LSK cells, which is consistent with a recent finding that Sirtuin 1 KO mice exhibit premature aging of BM. We also asked how LSK cells successfully maintained their homeostasis in BM during repetitive G spikes. To address this question, we examined the role of parasympathetic nervous system. For this, we injected G or saline for 7 days intoα7 nicotinic acetylcholine receptor knockout (chrna7 KO) mice. We found that G injected chrna7 KO mice BM also showed premature aging-like phenotype. Our study suggests that repetitive glucose/triglyceride spikes in T2DM induce premature aging of BM, very rapidly. Our study also suggests a new role of parasympathetic nervous system for the maintenance of BM homeostasis under metabolic fluctuation.

P01-6Visceral adipose t issue potent iates cardiac dysfunction in aging and obesityDaigo Sawaki1),�Gabor�Czibik1),� Costin� Radu1),� Julien�Ternacle1),�Corneliu�Henegar1),� Takehiko�Yoshimitsu2),�Geneviève�Derumeaux1)�1）INSERM U955, University of Paris East Créteil, 2）Laboratory of Synthetic Organic and Medicinal Chemistry, Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama UniversityBackground: Aging and obesity induces cardiac structural remodeling and dysfunction, suggesting predisposition to the development of heart failure with preserved ejection fraction (HFpEF). Adipose tissue is affected by a remarkable amount of structural and cellular remodeling throughout aging and obesity. Adiposity and HFpEF risk have demonstrated strong association in clinical studies, however, the underlying mechanism relating them remains elusive.Methods; Results: To elucidate the role of visceral adipose tissue (VAT) in age- or obesity-related cardiac dysfunction, we employed VAT removal or sham operation on aged mice (11 months old) and mice fed on high-fat diet (HFD for 16 weeks, until 9 months of age). Neither models demonstrated any difference in systemic adiposity and metabolic function such as glucose and insulin tolerance tests. Interestingly though, ventricular myocardium showed significantly reduced interstitial fibrosis, fibroblast area and Smad3 phosphorylation after VAT removal in both aged and obese mice. VAT removal also resulted in a dramatic restoration of systolic and diastolic function as assessed by strain-rate echocardiography and in vivo hemodynamics. Intriguingly, this restoration was coupled with selective cardiac fibroblast senescence in myocardium. Plasma analysis showed significantly lower levels of fibrotic factors such as osteopontin (OPN), TGFb1 and leptin after VAT removal. Furthermore, we confirmed that OPN was specifically induced in VAT during aging. OPN deficiency as well as a pharmacological OPN inhibitor showed preserved myocardial structure and function, suggesting that OPN is a key molecule inducing age-related cardiac dysfunction. Conclusion: These findings established visceral adipose tissue and OPN induction as a fundamental basis for cardiac fibrosis and dysfunction mediated by secreted fibrotic factors. Our study also unveiled cardiac fibroblast senescence as an important mechanism contributing to myocardial fibrosis restoration. These novel insights open interesting perspectives in the prevention or treatment of age- and obesity-related cardiac dysfunction, comprising a large portion of HFpEF.

P01-7Osteoprotegerin as a protective factor in the aging heartToshihiro Tsuruda1),�Yujiro�Asada1),�Kazuo�Kitamura1),�Kinta�Hatakeyama2),�Nobuyuki�Udagawa3),�Yoko�Sekita-Hatakeyama2),�Johji�Kato1)

1）University of Miyazaki, 2）Nara Medical University, 3）Matsumoto Dental UniversityBackground: Dysregulation of receptor activator of nuclear factor-kappa b ligand and osteoprotegerin (OPG) is associated with postmenopausal osteoporosis and rheumatoid arthritis. However, their pathophysiological role in changes in cardiac structure and function with aging remains to be elucidated. Methods and results: We conducted experiments using 2.5- and 12-month-old OPG-/- mice and age-matched wild type mice and compared the morphology and function of the left ventricle (LV). Both 2.5- and 12-month-old OPG-/- mice showed a higher systolic blood pressure than did age-matched wild type mice (122±2 vs.103±1 mmHg at 2.5 months old, 116±2 vs.101±1 mmHg at 12 months old, p=0.001, means±SEM), as well as a greater heart weight/body weight ratio (6.3±0.09 vs.4.8±0.09 mg/g at 2.5 months old, 8.1±0.64 vs.4.7±0.11 mg/g at 12 months old, p<0.001). Transthoracic echocardiograms revealed that 12-month-old OPG-/- mice displayed a significantly larger LV chamber size (3.4±0.3 vs.2.1±0.1 mm at diastole, p=0.003) with thinner in the wall thickness (0.8±0.03 vs.1.0±0.05 mm at diastole, p=0.0019) than age-matched wild type mice, resulting in a decrease in systolic fractional shortening (44±3 vs.67±2%, p<0.001). The observed morphological differences were accompanied by increase in numbers of apoptotic cells (3.1±1.0 vs.0.1±0.1/40,000 nuclei, p=0.0256) and decrease in area of interstitial fibrosis (0.08±0.02 vs.0.15±0.03%, p=0.0435) in the LV of 12-month-old OPG-/- mice. Correspondingly, OPG siRNA induced the expressions of TRAIL and cleaved caspase-3 in cultured cardiac myocytes. Furthermore, 12-month-old OPG-/- mice display activations of matrix metalloproteinase (MMP)-2, tissue inhibitors of MMP-1, -2 with the inactivation of procollagen alpha1 synthesis, and more phosphorylation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) than age-matched wild type mice. Intraperitoneal administration of 10 mg/kg recombinant human OPG to 12-month-old OPG-/- mice for 28 days led to partial improvement of LV systolic fractional shortening (before 32±4% and after treatment 41±1%, p=0.0374) without affecting systemic blood pressure. Conclusion: These results suggest that OPG plays a role in preserving myocardial structure and function with aging through reduced apoptosis and preserved matrix structure.

P01-8Endothelial cell senescence is involved in the progression of atherosclerosisSakiko Honda1),�Koji� Ikeda2),�Atsushi�Hoshino1),� Ryota�Urata1),�Sinichiro�Yagi1),�Satoaki�Matoba1)

1）Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 2）Laboratory of Clinical Pharmaceutical Science, Kobe Pharmaceutical University[BACKGROUND]Vascular aging has been considered to be associated with age-related organ dysfunction. Previous studies using aged mice revealed that aging caused endothelial cell (EC) dysfunction; however, it remains unclear whether EC senescence plays causative roles in age-related disease such as atherosclerosis.[METHOD AND RESULT]We embarked on generating mice in which ECs are specifically senescent. TRF2 is a teromere binding protein plays a critical role in the protection of chromosome ends. Inhibition of TRF2 by overexpressing TRF2-dominant negative (DN) induces DNA damage, leading to premature senescence. We confirmed that overexpression of TRF2DN caused premature senescence in ECs. Accordingly, we generated transgenic mice that overexpress TRF2DN under the control of the TIE2 promoter (TIE2-TRF2DN-Tg). TRF2DN-Tg and ApoE-KO/TRF2DN-Tg mice were fed with a high cholesterol diet, and the aortic valve annulus and thoracoabdominal aorta were extracted to assess atherosclerotic legion by using oil red-O staining after 2-week of high cholesterol diet. ApoE-KO/TRF2DN-Tg mice showed exacerbated atherosclerotic lesion in this early stage of atherosclerosis. To investigate the molecular mechanisms, we examined the foam cell formation of macrophages treated with conditioned medium of senescent ECs, but there was no difference in the conditioned medium derived from young or aged ECs. We then analyzed the inflammatory response in ECs. Young or senescent ECs were stimulated with TNF-α or IL-1β, and induction of VCAM-1, ICAM-1, and E-selectin expression was analyzed. Induction of these adhesion molecules in response to low-grade inflammatory stimuli was enhanced in senescent ECs. [CONCLUSION]Here, we revealed a causative role of EC senescence in the progression of atherosclerosis in vivo. Our in vitro studies strongly suggested that senescent ECs were highly sensitive to low-grade inflammatory stimuli, which potentially caused the exacerbated atherosclerosis in EC-specific progeroid mice. Although detailed underlying mechanisms remain to be elucidated, our findings revealed that EC senescence is a bona fide risk for atherosclerosis, and thus senescent ECs are attracting pharmacotherpeutic targets to prevent and/or treat atherosclerotic disease.

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P02-1Molecular bases for the sex difference in aortic aneurysm formation in Marfan syndromeQing Liu1),� Hiroshi�Akazawa1,2),� Hiroki� Yagi2),� Yusuke�Adachi1),�Akiko�Saga-Kamo2),�Masahiko�Umei1),�Hiroshi�Matsunaga1), � Hiroshi� Kadowaki1), � Ryo�Matsuoka1),�Kazutaka�Ueda2),�Norifumi�Takeda1,2),�Issei�Komuro1,2)

1）Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 2）Department of Cardiovascular Medicine, The University of Tokyo Hospital

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with genetic predisposition to aortic aneurysms and dissection. Epidemiologic evidence suggests that males are at higher risk for increased aortic growth or aortic events such as aortic dissection and/or prophylactic surgery than females, although MFS is equally prevalent in males and females. However, it is not clear what determines the sex differences in aortic phenotypes. Our study attempted to examine the influence of sex hormones on aortic aneurysm formation in mice heterozygous for the Fbn1 allele encoding a missense mutation (Fbn1C1039G/+, the most common class of mutation in MFS). We first observed that male MFS mice developed aortic dilatation more progressively than female MFS mice. To examine the effects of sex hormones on aortic aneurysm formation, we next performed ovariectomy (OVX) and orchidectomy (ORX) in female and male MFS mice, respectively. OVX did not induce a significant change in aorta diameters and fragmentation of elastin fibers, as revealed by Van Gieson staining, indicating that estrogen is not protective against aortic aneurysm formation in female MFS mice. In contrast, ORX significantly prevented progression of aortic dilatation, indicating that androgen is detrimental to aortic aneurysm formation in male MFS mice. These results suggest that, in Fbn1C1039G/+ MFS mouse, sex hormones may contribute to the sex difference in aortic phenotypes, and androgen may increase the susceptibility of males to aortic complications.

P02-2The integr ity of extracel lular matr ix af fects transduction of mechanical stress in the aortic wallThang Quoc Bui1),�Yoshito�Yamashiro2),�Seung�Jae�Shin2),�Hiroaki�Sakamoto1),�Yuji�Hiramatsu1),�Hiromi�Yanagisawa2)

1）Department of Cardiovascular Surgery, University of Tsukuba, 2）Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of TsukubaBackground:The extracellular matrix (ECM) plays a critical role in the maintenance of aortic wall. Hypertension is a common risk factor foraorticdiseases and induces remodeling of the aortic wall;however, the mechanism of aortic wall remodeling in response to mechanical stress and how ECMaffects these processesare not completely understood. The mutant mice with defective elastic fiber-associated ECM proteins such as elastin (Eln) and fibulin-5 (Fbln5) exhibit reduced or disorganized elastic fibers, respectively and develop narrowing of the aorta (in Eln+/-) or tortuous elongated aorta (in Fbln5-/-). In addition, we have previously found that matricellular protein,thrombospondin-1 (Thbs1), which mediates cell-matrix interactions,was increasedby mechanical stress. Therefore, we hypothesized that integrity of ECM affects aortic wall remodeling response to mechanical stress. Aim: To examine the responses of aorta with compromised ECM to mechanical stress in vivo, we performed Transverse Aortic Constriction (TAC) in Eln+/-, Fbln5+/-and Thbs1-/-mice and evaluated the changes in the aortic wall by histological, morphological and molecular biological approaches. Results:Eight weeks-old male mice were used for TAC and evaluated at 5 weeks post-operation. Although TAC induced aortic wall thickening in all groups,Thbs1-/- aortas showed significantly thicker aortic wall compared with wild-type aortas. Induction of mechanical stress was not sufficient to cause aortic diseases, including aneurysm and dissection,in wild-type, Eln+/- or Fbln5+/- mice.In contrast, Thbs1-/- mice showed decreased survival after TAC (46.1%) compared with wild-type mice (100%), and the necropsy and histological analyses revealed aortic dissectionwith abnormal collagen deposition and elastic fiber fragmentation. Conclusions:Maladaptive remodeling of the aortic wall was most likely caused by altered mechanotransductiondue to loss ofThbs1. Further analysis is required to elucidate the role of Thbs1 in aortic wall remodeling under the me ch a n i c a l s t r e s s a nd i t s p o s s i b l e c o n t r i b u t i o n t o cardiovasculardiseases.

P02-3Cell-specific function of fibulin-4 in progression of ascending aortic aneurysm in miceCaroline�Antunes�Lino1),�Yoshito Yamashiro2),�Biu�Quoc�Thang3),�Juliano�Vilela�Alves4),�Shin�Seung�Jae2),�Hiromi�Yanagisawa2)

1）Department of Anatomy, University of Sao Paulo, Institute of Biomedical Sciences, Sao Paulo, Brazil, 2）Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan, 3）Department of Cardiovascular Surgery, University of Tsukuba, Ibaraki, Japan, 4）Department of Pharmacology, University of Sao Paulo, Ribeirao Preto Medical School, Sao Paulo, BrazilBackground: Fibulin-4 (Fbln4) is an extracellular matrix (ECM) protein mainly expressed in blood vessels and is associated with microfibrils surrounding elastic fibers. We previously showed that smooth muscle cell (SMC)-specific deletion of fibulin-4 (Fbln4SMKO; SMKO) in vivo led to ascending aortic aneurysms. In SMKO aortas, the response to mechanical stress was elevated with upregulation of mechanosensitive protein, Thrombospondin-1 (Thbs1), in both endothelial cells (ECs) and SMCs. However, the function of Fbln4 in ECs and how it affects mechanosensing of the aortic wall are unknown. Aim: This study aims to elucidate the role of endothelial Fbln4 in the context of aortic aneurysm formation. Methods and Results: EC-specific knockout mice for Fbln4 (Fbln4ECKO; ECKO) were generated and crossed with SMKO mice to generate double knockout mice (Fbln4SMKO/ECKO; DKO). Animals were sacrificed at 2 months of age and morphological analysis was performed. ECKO mice appeared normal and did not develop any aortic aneurysms. In contrast, DKO mice showed large aneurysms expanding from the aortic root to the aortic arch, demonstrating the exacerbated aneurysm phenotype compared to SMKO mice. The blood flow velocity measured by echocardiogram revealed a turbulent flow in DKO aorta as evidenced by the concomitant antegrade and retrograde flow velocity. DKO mice showed a significant increase in heart weight/body weight ratio over control, indicating the development of cardiac hypertrophy. Furthermore, DKO aorta presented severe tortuosity in the descending aorta with upregulation of Thbs1 and formation of descending aortic aneurysms at 4-months of age. Conclusion: Deletion of Fbln4 in ECs and SMCs result in severe aortic aneurysms with disturbed flow and cardiac hypertrophy. These data indicate that Fbln4 deficiency may lead to endothelial dysfunction and progression of aortic aneurysms.

P02-4Role of PAR1-Egr1 in the initiation of thoracic aortic aneurysmSeungjae Shin1),�Yoshito�Yamashiro1),�Hiromi�Yanagisawa1)

1）Tsukuba universityBackground: Remodeling of the extracellular matrix (ECM) by matrix proteases plays an important role in cardiovascular homeostasis. Protease activated receptor 1 (PAR1) is a member of G-protein-coupled receptors (GPCRs) and is activated by a variety of proteases, including thrombin, matrix metalloproteinase (MMP)-1, 2 and 9. Thus, PAR1 enables cells to respond to the changes in the extracellular microenvironment. We have previously reported that loss of ECM protein fibulin-4 (Fbln4) in vascular smooth muscle cells (SMCs) in vivo (termed SMKO) led to ascending aortic aneurysms. Transcription factor, early growth response 1 (Egr1), which responds to mechanical stress, was markedly upregulated in SMKO aortas prior to aneurysm formation. However, the relationship between loss of Fbln4 and upregulation of Egr1, and whether proteases are involved in aneurysm initiation are unknown. Results: To identify the upstream signaling(s) of Egr1, we performed microarray analysis between control and SMKO aortas. Mmp2, Notch3 and Piezo2, which are known as a mechanosensor, were highly expressed in SMKO aorta at postnatal day 90 (P90). In vitro stretch assays (1.0Hz, 20% strain, 8hr or 20hr) using rat vascular SMKOs induced secretion of proteases, including Mmp2, carboxypeptidase E and complement C1r. Bioinformatic analysis using Ingenuity Pathway Analysis (IPA) revealed that cell surface receptors such PAR1, PAR4, and serum response factor accessory protein 2 (ELK3) are candidates for potential Egr1-interacting proteins. Interestingly, similar to Egr1, PAR1 transcripts were increased in SMKO aortas as early as P1 and PAR1 protein level was highly increased in the ascending aorta of SMKO at P30. Conclusion: These data indicate the possibility that mechanical stimuli in the SMKO aorta induce activation of PAR1 via secretion of proteases and upregulate mehanosensitive transcription factor Egr1. Further analysis on the relationship between PAR-1 activation and Egr-1 as well as the causal effect of PAR-1-Egr1 pathway in the initiation of aortic aneurysm needs to be examined.


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P02-5The role of Syk in pathogenesis of aortic dissectionYouhei Hashimoto1),�Hiroki�Aoki2),�Satoko�Ohno1),�Makiko�hayashi1),�Aya�Furusho1),� Ryouhei�Majima1),� Yoshihiro�Fukumoto1)

1）Department of Internal Medicine, Division of Cardiovascular Medicine, Kurume University School of Medicine, 2）Cardiovascular Research Institute

Aortic dissection (AD) is a serious clinical condition with unknown etiology that frequently results in fatal outcome. Recent studies showed essential role of inflammatory response both in AD and aortic aneurysm (AA), another destructive aortic disease. However, molecular pathogenesis of AD is much less characterized compared to AA. The difference of the molecular pathogenesis between AD and AA is also unclear. Very recently, we reported that Syk, a tyrosine kinase that regulates the differentiation and activation of inflammatory cells, promotes AA formation in a mouse model. In the current study, we investigated the role of Syk in AD. A mouse AD model was created by continuous infusion of beta-aminopropionitrile and angiotensin II (BAPN+AngII), which caused AD in almost all of the mice in 2 weeks. Immunohistochemical staining for activated (phosphorylated) Syk (pSyk) revealed that Syk was inactive in normal mouse aorta, but was strongly activated in the aortic walls after AD development. Double immunofluorescence staining for pSyk and smooth muscle alpha actin showed Syk was active not only in the infiltrating inflammatory cells, but also in smooth muscle cells in AD tissue. We examined the significance of Syk activation in AD by treating mice with fostamatinib, a specific Syk inhibitor, before and during BAPN+AngII infusion. Unexpectedly, the fostamatinib-treated group developed more severe AD compared to the vehicle-treated group. Furthermore, administration of fostamatinib resulted in significantly higher mortality due to AD rupture as shown by Kaplan-Meier analysis. These findings uncovered the previously unrecognized role of Syk for the aortic tissue protection, and suggested the fundamentally different disease mechanisms of AD and AA.

P02-6Involvement of Focal adhesion kinase in pathogenesis of aortic dissectionRyohei Majima1),�Hiroki�Aoki2),�Yohei�Hashimoto1),�Sohei�Ito1),�Makiko�Hayashi1),�Satoko�Ohno-Urabe1),�Yoshihiro�Hukumoto1)

1）Division of Cardiovascular Medicine, Department of Internal Medicine, Kurume University School of Medicine, 2）Cardiovascular Research Institute, Kurume UniverisityAortic dissection (AD) is a fatal disease in which the aortic media suddenly fails. AD involving the ascending aorta (Stanford type A) is particularly at the high risk of fatal outcome, for which surgical replacement of the damaged aorta with an artifical graft is highly recommended. Currently, no pharmacological therapy is available to limit the destruction of the aortic walls in AD, because its molecular pathogenesis is unknown. Recently, we discovered that MRTF-A, a mechanosensitive transcriptional regulator that is under the control of actin dynamics, is critically involved in AD pathogenesis. This discovery prompted us to investigate the role of focal adhesion kinase (FAK), which transduces the mechanostress to the actin dynamics, in AD pathogenesis. We used a mouse model of AD that was created by continuous infusion of beta-aminopropionitrile, a collagen crosslink inhibitor, and angiotensin II (BAPN+AngII), which caused AD in all mice with approximately 60% of mortality due to the AD rupture within 2 weeks. Western blot analysis for activated (phosphorylated) FAK (pFAK) showed that BAPN+AngII administration resulted in the activation of FAK in the aortic tissue before AD development. Immunohistochemical staining for pFAK also revealed that FAK was strongly activated in the aortic walls after BAPN+AngII infusion. Double immunofluorescence staining for pFAK and smooth muscle alpha-actin showed that FAK was activated both in smooth muscle cells and in non-smooth muscle cells after the BAPN + AngII challenge. Significantly, administration of PND-1186, an orally available FAK inhibitor, resulted in the significant reduction in the severity of AD especially in the aortic arch including the ascending aorta. Furthermore, the mortality was improved from 63.1% to 20.0% by PND-1186 administration. These findings demonstrated that FAK plays a central role in AD pathogenesis, possibly by transducing the pathological stress to the tissue destructive response in the aortic walls. We propose that FAK is a potential therapeutic target to limit the fatal destruction of aortic walls in AD.

P02-7Impact of CD44 on the development of thoracic aortic aneurysm and dissection in miceOmer Hatipoglu1),�Toru�Miyoshi1),�Kazufumi�Nakamura1),�Masashi�Yoshida1),�Hiroshi�Ito1)

1）Okayama UniversityIntroduction: Thoracic aortic aneurysm dissection (TAAD) is a life-threatening vascular disease without effective pharmaceutical therapy. However, the underlying pathological mechanisms of TADD remain elusive. Previous studies reported that inflammatory cell CD44 plays an important role in leukocyte recruitment to sites of inflammation. In this study, we investigated the role of CD44 in the development of TAAD in murine model.Methods: For induction of TAAD, CD44 deficiency (CD44-/-) or wild-type (WT) mice were infused with β-aminopropionitr i le monofumarate (BAPN, a lysyl oxidase inhibitor, 100ug/kg per minute) and angiotensin II (Ang II, 1 ug/kg per minute) simultaneously for 7 days via osmotic mini pumps.Results: An increase in systolic blood pressure was observed equivalently in both groups. At 7 days, BAPN/Ang II infusion developed TAAD in 69% of WT mice with 16 % dying from rupture of the aortic dissection, but in 26% of CD44-/- mice with 10 % dying (p<0.01 in the incidence of TAAD). After 24 hours BAPN/Ang II infusion, neutrophil accumulation observed in the adventitia of the thoracic aorta in CD44-/- mice was significantly lower than that in WT mice (1.61%, vs. 5.65%, Ly6B.2-positive area, p<0.01). Quantitative real time PCR showed that the increase in IL-6 and TNF alpha in thoracic aorta in the CD44-/- mice was significantly reduced compared to WT mice. Zymography showed that activity of matrix metalloproteinase-9 in thoracic aorta in the CD44-/- mice were significantly decreased compared to WT mice. Finally, neutrophil migration across TNF-alpha activated endothelial cell monolayers was evaluated. Migration of neutrophils derived from CD44-/- mice was significantly decreased by 18% compared to that of neutrophils derived from WT mice.Conclusions: Our results suggest that CD44 contributes to the development of TAAD via regulation of neutrophils infiltration at an early stage. Targeting CD44 may represent a strategy for inhibiting the formation of TAAD.

P02-8Angiotensin receptor-Neprilysin Inhibition Attenuates Aortic Aneurysm in MiceKouhei Tashiro1),�Takashi�Kuwano1),�Yasunori�Suematsu1),�Shin-ichiro�Miura1)

1）Fukuoka University School of MedicineIntroduction: Angiotensin-neprilysin inhibitor (ARNI) consists of the neprilysin inhibitor sacubitril and the angiotensin receptor blocker valsartan, and this new combination drug is superior to enalapril at reducing the cardiovascular death for patients with heart failure. Hence, combined inhibition has some additive effect and it is likely to be a useful drug for the other cardiovascular disease as well. Angiotensin II (Ang II) modulates progression of aortic aneurysm, but pharmaceutical intervention to renin-angiotensin system for the prevention of aortic aneurysm is not well established. In this study, we investigated whether ARNI has an effect against mouse model of aortic aneurysm. Method: In Ang II-induced (1.44 mg/kg/day) aortic aneurysm models, oral varsartan (30 mg/kg/day; VAL group) or varsartan/sacubitril (30 mg/kg/day varsartan, and 30 mg/kg/day sacubitril; ARNI group) or corn oil (Vehicle group) were given for the same period as the Ang II infusion. Blood pressure was measured by weekly and transthoracic cardiac echography. After treatment, aortic aneurysm development was compared among groups. Results: Ang II infusion resulted in a significant increase in blood pressure only in vehicle group. No difference in blood pressure was observed throughout the experiment between VAL group and ARNI group. Fatal aortic rupture was occurred by 17% during treatment period, but there was no significant difference among groups. After 4 weeks of treatment, mice received ARNI had a significantly decreased incidence of non-fatal aortic aneurysm (Vehicle group: 100%, VAL group: 78%, ARNI group: 33%). ARNI treatment also suppressed the development of aortic aneurysm with significant reduction of maximum diameter of abdominal aorta (Vehicle group: 2.2±0.7mm, VAL group: 1.8±0.7mm, ARNI group: 1.8±0.4mm, p=0.003 for Vehicle vs ARNI, p=0.03 for VAL vs ARNI). Conclusion: Combined angiotensin receptor blockade and neprilysin inhibition had a suppressive effect for aortic aneurysm formation in a mouse model of aortic aneurysm.

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P03-4Stabilization of symptomatic carotid atherosclerotic plaques by statins: A pathological analysisTakao Konishi1),�Shinya�Tanaka1)

1）Department of Cancer Pathology, Hokkaido University, Graduate School of Medicine

Background and Purpose: Human and animal studies have revealed a stabilization of atherosclerotic plaques by statins. However, the stabilization of human carotid plaques has not been thoroughly described pathologically. This analysis explored the relationship between stat in therapy and p laque stab i l i ty in carot id endarterectomy (CEA) specimens.Methods: We analyzed specimens harvested between May 2015 and February 2017, from 79 consecutive patients presenting with >70% carotid artery stenoses, of whom 66 were untreated (group 1) and 13 treated (group 2) with a statin. Immunohistochemistry was performed, using an endothelial specific antibody to CD31, CD34 and platelet derived growth factor receptor-β. Results: The prevalence of plaque ruptures (P=0.009), lumen thrombi (P=0.009), inflammatory cells (P=0.008), intraplaque hemorrhages (P=0.030) and intraplaque microvessels (P<0.001) was significantly lower in group 2 than in group 1. Among 66 patients presenting with strokes and infarct sizes >1.0 cm3 on magnetic resonance imaging, the mean infarct volume was significantly smaller (P = 0.031) in group 2 (4.2 ± 2.5 cm3) than in group 1 (8.2 ± 7.1 cm3). The difference in mean concentration of low-density lipoprotein cholesterol between groups 1 (121 ± 32 mg/dl) and 2 (105 ± 37 mg/dl) was non-significant (P=0.118).Conclusions: This analysis of plaques harvested from patients undergoing CEA suggests that statin therapy mitigates the plaque instability, which, in patients presenting with strokes, might decrease infarct volume.

P03-130-day morbidity and mortality of patients with acute coronary syndromeThatsaphone Somsaart1),�Pheng�Sackda1)

1）Setthathirath HospitalBackground: Coronary artery disease (CAD) is also one of the major causes of morbidity and mortality in Lao People’s Democratic Republic (Lao PDR). However, the morbidity and mortality after presenting for treatment is unknown in Lao PDR. Morbidity and mortality may be worse in Lao PDR due to a lack of facilities for appropriate diagnosis and treatment. For example, certain laboratory tests are not available, and there is no coronary revascularization in the country. Additionally, due to poverty and low education, patients are often not aware that they have CAD, are unable to access or afford health care, and present late in the disease course. Purpose: This study was undertaken to describe the 30-day morbidity and mortality of patients with acute coronary syndrome (ACS) after presenting to definitive medical care at Mahosot Hospital, Vientiane, Lao PDR. Method: From February 2016 to June 2017, all patients who were admitted to the cardiovascular ward with a diagnosis of ACS at Mahosot Hospital were enrolled in this prospective observational study. Researchers interviewed each patient and reviewed the medical charts during the patients’ hospital stays and made follow-up phone calls and/or out-patient department visit during the 30 days after discharge. Results: Fifty-three patients were enrolled to the study. All patients were aged 37 to 87 years, with a mean age of 63 years. Seventy percent of patients had ST elevation myocardial infarction (STEMI). Forty percent of patients had acute heart failure (AHF). Of the patients with AHF, fifty-two percent had cardiogenic shock, all were diagnosed with STEMI and seven died in hospital. Three patients were re-admitted (one with heart failure, one with supraventricular tachycardia, and one with recurrent myocardial infarction). Conclusion: This study demonstrates the morbidity and mortality of patients with ACS in Vientiane, Lao PDR after presentation to definitive medical care. Many patients with ACS had AHF. Over half of patients with ACS had cardiogenic shock, especially patients with ST elevation myocardial infarction. Most patients with cardiogenic shock died in hospital. To improve morbidity and mortality, it is essential to improve hospital care and prevent AHF and cardiogenic shock. Keywords: Acute coronary syndrome, morbidity, mortality.

P03-2STEMI PATIENT IN TYPE C HOSPITAL : TREAT OR NOT TO TREAT ?Lianita Gumdani1),�Hartogu�Aprico�Humisar�Panjaitan1),�Arif�Qadhafy1),�Kabul�Priyantoro1)

1）Mekarsari Hospital

ST-elevated myocardial infarction (STEMI) caused by complete occlusion of an epicardial coronary artery. Revascularization is mandatory in saving infark related teritory. In JKN (Indonesian National Health Insurance) era, there are some limitacy of type-C hospital in managing STEMI patient and there is no report of primary-PCI. Fibrinolytic become an option with onset less than 12 hours. To describe the management of STEMI patient in type-C hospital. We conducted a descriptive study, data taken from medical records between April 2014- January 2018. STEMI was diagnose from symptoms and ECG pattern. There were 48 STEMI patients with onset less than 12 hours (42 males and 6 females). The mean age was 52 years old with the oldest 74 years old and the youngest 30 years old. All of them 48 (100%) patients were covered by JKN and received fibrinolytic therapy with door-to-needle mean time was 28,87 minutes and in-hospital mortality rate was 8,3% (4 patients). The most risk factor of STEMI is hypertension (47,9%). There were 12 patients came with onset more than 12 hours. Type-C hospital during JKN era can contribute to optimal management of STEMI patient by delivering fibrinolytic theraphy. Hypertension was still the most common risk factor for STEMI patients.Keywords :STEMI, Fibrinolytic, Type-C Hospital

P03-3A o r t i c v a l v e c a l c i f i c a t i o n a n d c o r o n a r y atherosclerosis in familial hypercholesterolemia patientsEri Yamamoto1),� Kayoko� Sato1),� Haruki� Sekiguchi1),�Yasutaka� Imamura1),�Hidekimi�Nomura1),�Akiko�Sakai1),�Nobuhisa�Hagiwara1)

1）Department of Cardiology, Tokyo Women's Medical University

Introduction: Familial hypercholesterolemia (FH) is caused by mutations of FH genes. On the other hand, aortic valve calcification (AVC) is associated with an elevated risk of cardiovascular events.Objectives: To investigate the association between AVC and coronary plaque morphology related with gene mutation.Methods: We enrolled 42 heterozygous FH (heFH) and 31 hypercholesterolemia (DL) patients and evaluated 320 detector row CT, ABI and PWV. The genotypes were analyzed using Illumina Mi-seq sequencer.Results: AVC was detected more frequently and AVC score was higher in heFH than in DL (p<0.01, respectively). AVC score was significantly associated with coronary artery plaque score (CAPS; p<0.01). In heFH, 23 patients (55%) had gene mutations. The gene mutation of LDLR, APOB, and PCSK9 was observed in each of 10 (37%), 2 (7%), and 11 (41%) heFH patients. The double gene mutation of LDLR and PCSK-9 was observed in 4 (15%) of heFH. FH patinets with LDLR or PCSK9 mutation had higher AVC score (P<0.05) and CAPS(P<0.05). Furthermore, some of heFH had the gene mutation of ABCG5 / ABCG8 (8, 30.7%), or APOA5 (6, 23.1%). Coronary atherosclerosis was more developed in heFH with polygenetic gene mutation.Conclusions: The AVC is developed and related to coronary plaque morphology using computed tomography in heFH, especially with LDLR or PCSK9 mutation. The genetic diagnosis of heFH is very helpful to assess severity of coronary atherosclerosis and to determine the treatment strategy for preventing the future cardiovascular events.


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P03-5Validation of OCT findings in vivo plaque histology obtained by directional coronary atherectomyHiroshi Okamoto1),� Teruyoshi�Kume1),� Koichiro� Imai1),�Ryotaro�Yamada1),�Yoji�Neishi1),�Shiro�Uemura1

1）Kawasaki medical schoolBackground: The comparison between histological characteristics and optical coherence tomography (OCT) / optical frequency domain imaging (OFDI) findings has not fully examined among stable angina patients. Coronary plaque component in vivo could be obtained by directional coronary atherectomy (DCA) and evaluated in histological examination. The purpose of this study was to evaluate the relationship between histological plaque characteristics obtained by DCA and OCT findings in stable angina patients. Methods: A total of 40 specimens from 17 stable angina patients underwent OFDI guide PCI using DCA was analyzed in this study. Routine OFDI observations were performed among pre DCA and each post DCA. OFDI images were recorded from the distal to the proximal arterial segments to include the stenosis lesion of the artery at a pull-back speed of 40 mm/s (160 frames/s). The cutting zone of pre DCA OFDI image was classified into two categories; homogeneous or heterogeneous. Histological specimens obtained by DCA were divided into 2 groups, homogeneous or heterogeneous in OFDI images. In matched sections, specimens were assessed histological findings. Histological specimens were fixed in 10% formalin and embedded in paraffin using standard protocols. Each specimen was entirely cut in 3 μ m serial sections and stained with hematoxylin, e o s i n and p i c ro s i r i u s r ed s t a i n i ngs and examined on immunohistochemistry using primary monoclonal antibodies anti-glycophorin A for the detection of thrombosis, respectively. Result: A total of 40 specimens from 17 patients was obtained by DCA. 14 specimens were classified as homogeneous plaques in OFDI image and 26 specimens were classified heterogeneous plaques in OFDI image. All homogeneous plaques were composed of collagen-rich fibrous tissue (100%). Fifteen heterogeneous plaques (58%) were consisted of organized thrombosis. Six heterogeneous plaques (23%) were consisted of collagen-rich fibrous tissue. Conclusion: Heterogeneous plaque characteristics observed by OCT probably represent organized thrombus in culprit lesions of stable angina patient.

P03-6Influence of thrombin on the structure of platelet glycoprotein Ibα binding to von Willebrand factorShinichi Goto1,2),� Kengo�Ayabe2),� Hiroto� Yabushita2),�Hideki�Oka2),�Shinya�Goto2)

1）Keio University School of Medicine, 2）Tokai University School of MedicineIntroduction: Two binding sites for thrombin on glycoprotein (GP) Ibα (exosites I and II) have been reported based on biological experiments. These binding sites are important for regulating the platelet activation and thrombus formation. Though the thrombin binding site is close to VWF binding one, the influence of thrombin on the binding of GP Ibα to von Willebrand factor (VWF) is still unknown.Methods: We applied molecular dynamic (MD) simulation using the Chemistry at Harvard Macromolecular Mechanics (CHARMM) 36 force field to elucidate differences in stable binding structure of GP Ibα with VWF in the presence and absence of thrombin. The calculation was done on the Ultrafast Oakforest-PACS computer using the Nanoscale Molecular Dynamics (NAMD) package. The initial structures were obtained by aligning the crystal structure of thrombin bound to exosites I and II of GP Ibα to the stable binding structure of GP Ibα and VWF. The MD calculation were conducted in time step of 2 x 10-15 seconds until the binding of GP Ibα and VWF in-silico became stable. Results: After 300 ns (150 x 106 steps) of MD calculation, the binding structure was stabilized with root mean square distance deviation within 0.5Å. The bond between GP Ibα and thrombin was apparently unchanged. However, the binding of GP Ibα to VWF was markedly different when thrombin was bound to the exosites I and II of GP Ibα as compared with the binding when thrombin was absent. The relative position of GP Ibα to VWF in the presence of thrombin moved approximately 39Å away from the known GP Ibα binding site of VWF.Conclusions: The MD simulation predicted that the stable binding structure of GP Ibα and VWF is modified by thrombin. These results suggest an additional role of thrombin for controlling platelet adhesion at site of vessel injury under blood flow, which is mediated exclusively by GP Ibα binding with VWF.

P03-7Different impact of thrombin generation rate and enzymatic effects of thrombin on thrombus growth.Shinichi Goto1,2),� Kengo�Ayabe2),� Hiroto� Yabushita2),�Hideki�Oka2),�Shinya�Goto2)

1）Keio University School of Medicine, 2）Tokai University School of MedicineBackground: Differences in the effect of anti-thrombin therapy and inhibition of upstream of thrombin generation for the growth of thrombus formed under blood flow conditions are still to be elucidated.Method: A model of computer simulator implementing the function of blood flow, adhesive and activating functions of platelet cells, coagulation and fibrinolysis was developed. We defined the enzymatic effect of thrombin as conversion of fibrinogen to fibrin and platelet activation through protease activate receptor (PAR)-1. Thrombin generation rate was defined locally on activated platelet cell. The platelet thrombi in this system was defined as the region with 30% or more platelet cells were activated. To compare the impact of thrombin and thrombin generation, the following parameters were changed from 10% to 200% as compared to control (control rates were defined as the followings: fibrinogen to fibrin conversion rate as 0.1 (s-1), platelet activation rate by thrombin as 0.05 (s-1), and thrombin generation rate on activated platelet as 1 (s-1)). The sizes of platelet thrombi were calculated continuously in each 0.1 second after initiation of virtual endothelial injury. The biological validity of our computer simulation was previously established by comparative quantitative biological experiments with perfusion of whole blood on collagen fibrils under controlled blood flow conditions by parallel plate flow chamber. Results: The sizes of platelet thrombi increased over time of blood perfusion and stabilized after 30 seconds of calculation. The size of platelet thrombi reached to 0.0069 mm3 at control. Size of thrombi become smaller to 0.0049 mm3 and 0.0048 mm3 when the activity of thrombin or rate of thrombin generation decreased to 10% of control respectively. The size of thrombi of 0.0069 mm3 with 200% thrombin generation rate was same as the control while it increased to 0.0079 mm3 when the enzymatic activity of thrombin was 200%.Conclusion: Our computer simulation suggests different impact between the rate of thrombin generation and the enzymatic activity of thrombin on thrombus growth under blood flow condition.

P03-8Bacteroides inhibits atherosclerosis by regulating gut microbial LPS productionNaofumi Yoshida1),�Tomoya�Yamashita1),�Takuo�Emoto1),�Hikaru�Watanabe2),�Tomohiro�Hayashi1),�Tokiko�Tabata1),�Ken-ichi�Hirata1)

1）Kobe University Graduate School of Medicine, 2）Tokyo Institute of TechnologyBackground: Increasing evidence suggests a strong relationship between gut microbiota and cardiovascular diseases. We have previously reported that the abundance of genus Bacteroides is lower in patients with coronary artery disease (CAD) than in non-CAD patients with coronary risk factors. However, it still remains unclear which and how particular species within the genus Bacteroides contribute to development of atherosclerosis. Methods: We recruited 30 CAD patients and 30 non-CAD controls with coronary risk factors. We then compared the gut microbial profile using 16S rRNA gene sequencing analysis and quantitative polymerase chain reaction (qPCR) in feces to detect specific species with differential abundance. Subsequently, we used a mouse model to study the mechanisms underlying their relationship with atherosclerosis. Finally, we verified whether the mechanisms was observed in humans to bolster our data. Results: CAD patients demonstrated significantly lower abundance of Bacteroides vulgatus and Bacteroides dorei on fecal 16S rRNA gene sequencing analysis and qPCR. Gavage with B. vulgatus and B. dorei for 5 times per week at a dose of 2.5 × 109 cfu/100 μ L ameliorated endotoxemia, suppresed pro-inflammatory immune responses, and attenuated atherosclerotic lesion formation in atherosclerosis-prone mice. Changes in gut microbial composition followed by decreased expression of genes involved in LPS biosynthesis and fecal lipopolysaccharide levels in response to gavage with Bacteroides were observed. Furthermore, fecal lipopolysaccharide levels in CAD patients were significantly higher and negatively correlated with B. vulgatus and B. dorei abundance. Conclusions: We identified causal link between specific gut bacteria and atherosclerosis, propose fecal lipopolysaccharide levels as a novel dysbiosis marker for CAD, and suggest that Bacteroides treatment may prevent CAD.

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P04-4Direct interaction between adiponectin and oxidized LDL limits their action each otherAkemi Kakino1,2),� Yoshiko�Fujita1,3),� Sayaka�Horiuchi1),�Tatsuya�Sawamura1,3)

1）Department of Physiology, Shinshu University School of Medicine, 2）Institute for Biomedical Sciences, Shinshu University, 3）Research Center for Next Generation Medicine, Shinshu UniversityBackground: Adiponectin has anti-diabetic, anti-inflammatory, and anti-atherogenic properties. While the anti-diabetic role of adiponectin has been well defined through the study of its receptors, AdipoR1 and R2, the anti-atherogenic mechanism remains to be understood. On the other hand, it is generally recognized that oxidized LDL (oxLDL) can potentiate the formation of foam cells and induce the inflammatory responses that are believed to be crucial for atherogenesis.Aim: To elucidate how adiponectin exerts its anti-atherogenic action and how oxLDL affects adiponectin action.Methods and Results: At first, we examined oxLDL-adiponectin interaction. Adiponectin preferentially bound to oxLDL in ELISA, compared to native LDL. Adiponectin inhibited DiI-labeled oxLDL (DiI-oxLDL) uptake in COS-7 cells by the oxLDL receptors including LOX-1 and SR-A, while it did not affect the uptake of DiI-labeled native LDL uptake by the LDL receptor. These results indicate that adiponectin selectively blocks oxLDL binding. Adiponectin mostly inhibited DiI-oxLDL uptake by cultured human coronary artery endothelial cells (HCAECs) and THP-1 cells differentiated into macrophages as well. At second, to elucidate the significance of the complex formation, cellular responses to oxLDL were analyzed. Adipoenctin-oxLDL binding suppressed oxLDL-induced NF- κ B activation and ERK phosphorylation in CHO cells expressing LOX-1. In HCAECs, adiponectin suppressed the oxLDL-induced MCP-1 and ICAM-1 expression, and endothelin-1 secretion as well as oxLDL-induced signaling. Conversely, oxLDL suppressed the adiponectin-induced activation of AMP-activated protein kinase. Interestingly, T-cadherin, the receptor of adiponectin, strengthened the anti-oxLDL action of adiponectin, indicating receptor-level competition.Conclusions: Adiponectin protected endothelial cells from dysfunction by directly binding to oxLDL.

P04-1Comprehensive analysis of HIF-1a target genes in macrophage adaptation to the microenvironmentTakayuki Isagawa1),�Norihoko�Takeda2),�Hiroaki�Semba2),�Koji�Maemura1)

1）Department of Cardiovascular Medicine, Nagasaki University Graduate School of Biomedical Sciences, 2）Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo

Macrophages p l ay a cen t ra l r o l e i n the pa thogenes i s ofarteriosclerotic diseases such as angina and myocardial infarction. We previously showed that HIF-1a, the central mediator of hypoxic response, is involved in the inflammatory response of macrophage. We also revealed that HIF-1a plays important roles during the process of tissue remodeling in a mouse model of heart failure. While HIF-1a protein accumulation is elicited either by hypoxic stimulus or inflammatory signals including lipopolysaccharide (LPS), it is unclear whether hypoxia induced HIF-1a (hypoxia-HIF-1a) or LPS elicited HIF-1a (LPS-HIF-1a) has similar function in macrophage activation.. Therefore, we investigated the target genes of HIF-1a under hypoxic o r i n f l ammato ry c ond i t i o n s by Ch IP - S eq (Ch r oma t i n Immunoprecipitation with massive parallel DNA sequencing) analysis. While hypoxia-HIF-1a mostly induced its target genes at 4 hours, LPS-HIF-1a exhibited its target gene expression 24 hours after LPS stimulus. Furthermore, it was revealed that HIF-1a targeted glycolytic genes under both inflammatory and hypoxic conditions, and the number of LPS-HIF-1a target genes was significantly smaller than that of hypoxia-HIF-1a. Intriguingly, Pdk1 well-known as the mitochondria gate keeper gene was not induced under inflammatory condition. This result suggests that the glycolytic metabolism is different between hypoxic and inflammatory conditions. We are now investigating the physiological significance of such differences in HIF-1a regulated glycolytic metabolism between hypoxia and inflammation.

P04-2Telomerase Deficiency Prevents Vascular Smooth Muscle Cell Proliferation and Neointima FormationJun Aono1),�Mika�Hamaguchi1),�Chika�Suehiro1),�Kayo�Takahashi1),�Jun� Suzuki1),�Dennis�Bruemmer2),� Shuntaro� Ikeda1),�Osamu�Yamaguchi1)

1）Department of Cardiology, Pulmonology, Hypertension, and Nephrology, Ehime University Graduate School of Medicine, 2）Division of Cardiology, Department of Medicine, Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, UPMC and University of Pittsburgh School of MedicineINTRODUCTION- By maintaining the stability of telomeres, DNA-protein complexes that protect the ends of chromosomes, telomerase is rate limiting for tissue renewal and cell proliferation. In addition to this telomere maintenance function, mounting evidence suggests that the telomerase reverse transcriptase (TERT) exhibits a direct regulatory role in the activation of mitotic gene expression without apparent involvement of its well-established function in telomere homeostasis. In the present study, we investigated the role of TERT in vascular remodeling and the hypothesis that TERT regulates vascular smooth muscle cell (VSMC) proliferation through an epigenetic activation of an S Phase gene expression program. METHODS AND RESULTS- We first demonstrate high levels of TERT expression in replicating SMC of the developing neointima of human atherosclerosis. Studies in VSMC isolated from genetically engineered TERT-deficient mice with normal telomere length establish that TERT is necessary for cell proliferation. Conversely, overexpression of TERT induces cell proliferation without extending telomere length. Although TERT-deficient VSMC are not senescent, decreased proliferation is associated with stable and irreversible silencing of S phase gene expression, including target genes of the S Phase transcription factor E2F. Chromatin immunoprecipitation assays confirm that TERT is recruited to E2F target sites and promotes chromatin accessibility by facilitating the acquisition of permissive histone modifications. Finally, using a model of guide-wire induced arterial injury, we demonstrate these observations are applicable in vivo and identify decreased neointima formation in TERT-deficient mice. CONCLUSIONS- These data indicate a previously unrecognized role for TERT in the epigenetic regulation of proliferative gene expression in VSMC.

P04-3Glucocorticoid decreases uptake of Ac-LDL through suppression of JAK-STAT pathway in macrophagesRyota Hashimoto1),� Ryo�Kakigi1),� Kyoko�Nakamura1),� Yuki�Miyamoto2),�Hiroyuki�Daida2),�Takao�Okada1),�Youichi�Katoh2,3)

1）Department of Physiology, Juntendo University Faculty of Medicine, 2）Department of Cardiovascular Medicine, Juntendo University Graduate School of Medicine, 3）Juntendo University Faculty of International Liberal ArtsBackground: In the progressive process of atherosclerosis, atherosclerotic plaques are developed by accumulation of foam cells derived from macrophages via uptake of modified low-density lipoprotein (LDL). It is well known that CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified-LDL. Although glucocorticoid can increase occurrence and grades of atherosclerosis after a long-term treatment, mechanisms how glucocorticoid affects uptake of modified-LDL have not been fully elucidated.Methods and Results: We used mouse bone marrow macrophages and investigated effects of both a natural glucocorticoid (hydrocortisone, HC) and a synthetic glucocorticoid (dexamethasone, Dex) using flow cytometry and western blotting. One microM HC and 100 nM Dex decreased expression of CD36 but not CD204, resulting in decreased uptake of Ac-LDL in naïve macrophages. We next assessed the effects of glucocorticoid on activated macrophages using lipopolysaccharide (LPS). Interestingly, 1 microM HC and 100 nM Dex blocked enhanced uptake of Ac-LDL by 2 microgram/mL of LPS through suppression of CD204 expression but not CD36 expression. Two microgram/mL of LPS enhanced phosphorylation of signal transducer and activator of transcription-1 (STAT-1), and which was inhibited by pretreatment of 100 nM Dex. Moreover, inhibitors of janus tyrosine kinase (JAK)-STAT pathway, 1 microM Ruxolitinib and 1 microM Tofacitinib, blocked enhanced uptake of Ac-LDL by LPS through suppression of CD204 expression but not CD36 expression.These results suggest that glucocorticoid decreases uptake of Ac-LDL by at least 2 pathways; one is a reduction of CD36 expression in naïve macrophages and another is a blocking of enhanced CD204 expression through suppression of JAK-STAT pathway in LPS-activated macrophages. Conclusions: A short-term treatment of glucocorticoid on the initiation of atherosclerotic plaque may provide beneficial effects. Regulating CD204/SR-A activity through the JAK-STAT pathway will likely be an effective therapeutic tool to regulate various inflammatory diseases, such as septic shock and progressive atherosclerosis.


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P04-5DPP-4 inhibitors protect HUVECs from hypoxia-induced apoptosis and vascular barrier impairmentMasanori Hirose1), � Naoko� Hashimoto2), � Noritaka�Yamaguchi2),�Yoshio�Kobayashi1),�Hiroyuki�Takano2)

1）Department of Cardiovascular Medicine, Chiba University Graduate School of Medicine, 2）Department of Molecular Cardiovascular Pharmacology, Chiba University Graduate School of Pharmaceutical SciencesDipeptidyl peptidase-4 (DPP-4) inhibitors are oral anti-diabetic agents. Some protective effects of DPP-4 on cardiovascular disease have been described independently from glucose-lowing effect. However, the detailed mechanisms by which DPP-4 inhibitors exert on endothelial cells remain elusive. The purpose of this research was to determine the effects of DPP-4 inhibitor on endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured and exposed to hypoxia in the presence or absence of DPP-4 inhibitor. We confirmed that hypoxia leads to the injury of endothelial cells, which is represented by the increase in the number of apoptotic cells. DPP-4 inhibition had beneficial effects on HUVECs by inhibiting the apoptosis under hypoxia. The antiapoptotic effect of DPP-4 inhibitor was abolished by the pretreatment with CXCR4 antagonist or STAT3 inhibitor, indicating that SDF-1α/CXCR4/STAT3 pathway might be involved in the mechanisms of endothelial cell protection by DPP-4 inhibition. These results suggest that DPP-4 inhibitor has a potential for protecting vessels.Meanwhile, we confirmed that hypoxia leads to degradation of VE-cadherin and β-catenin complex, which plays a critical role in regulating vascular permeability and integrity. DPP-4 inhibitor plays a role in maintenance of VE-cadherin and endothelial adherens junction by suppressing cleavage of β-catenin. Caveolin-1 is an integral component of VE-cadherin and β-catenin complex in HUVECs. It was reported that soluble DPP-4 binds to caveolin-1 and then enhances the expression of NF-κB and TNF-α in THP-1 cells and monocytes. DPP-4 inhibitor inhibited hypoxia-induced translocation of NF-κB from cytoplasm to nucleus through decreasing TNF-α expression level. These findings suggest that DPP-4 inhibitor has beneficial effect on barrier impairment of HUVECs under hypoxic conditions. Furthermore, the tube formation assay showed that DPP-4 inhibitor significantly restored hypoxia-induced decrease in number of tubes by HUVECs. These results suggest that DPP-4 inhibitor protects HUVECs from hypoxia-induced barrier impairment.

P04-6Pericyte-specific deletion of Ninjurin1 induces abnormal vasa vasorum and intimal hyperplasia.Kiwamu Horiuchi1),�Akiho�Minoshima2),�Maki�Kabara1),�Taiki�Hayasaka1),�Naofumi�Takehara1),�Naoyuki�Hasebe2),�Jun-ichi�Kawabe1)

1）Department of Cardiovascular Regeneration and Innovation, Asahikawa medical university, 2）Department of Medicine, Division of Cardiovascular, Respiratory and Neurology, Asahikawa medical university

【Introduction】Growing vasa vasorum, microvessel in adventitia plays an important role in vascular remodeling in atherosclerotic lesion. Recently, we reported that Ninjurin1 (Ninj1) in pericytes (PCs) is crucial for maturation/stabilization of neovessels through PCs-endothelial cells (ECs) association. The purpose of this study is to clarify the role of Ninj1 in formation of vasa vasorum and vascular remodeling using a murine vascular injury model.【Methods】Deletion of Ninj1 gene in NG2-positive PCs was induced by tamoxifen(Tam)-treated NG2-CreER/Ninj1loxp mice (Ninj1KO, n=9). Controls (Ct) were prepared in either Tam-treated-NG2-CreER (Ct 1, n=4) or Tam-nontreated NG2-CreER/Ninj1loxp (Ct 2, n=5) mice. Femoral arteries were injured by insertion of coiled wire. After 4 weeks of surgery, circulating vessels were stained by venous injection of FITC-lection followed by paraformaldehyde fixation. Extracted femoral arteries were decolorized with CUBIC reagent and intimal hyperplasia, adventitial neovessels were estimated by immunostaining/histological analyses. 【Result】Wire injury induced intimal hyperplasia, as assessed by intima/media (I/M) ratio, and growth of microvessels in adventitia. Wire-injury-induced intimal hyperplasia in Ninj1KO were significantly enhanced compared to Cts. Formation of fragile microvessels associated with microbleeding in adventitia of injured vessels were enhanced in Ninj1KO. The number of macrophage in injured vascular walls were increased in Ninj1KO. 【Conclusion】Deletion of Ninj1 in microvessel PCs contributes to formation of immature microvessels in adventitia of injured vasculature. Enhanced neointimal formation in Ninj1KO may be mediated by inflammation through adventitial microbleeding.

P04-7Metabolic Stress Evokes Mitochondrial Dysfunction and Vascular Senescence via Mitochondrial FissionYoshihiro Uchikado1),�Yoshiyuki� Ikeda1),�Yuichi�Sasaki1),�Yuichi�Akasaki1),�Ohishi�Mitsuru1)

1）Cardiovascular Medicine and Hypertension, Graduate School of Medicine, Kagoshima University,Metabolic stress, such as oxidized low density lipoprotein (ox-LDL) and advanced glycation end products (AGE) cause mitochondrial dysfunction and evoke vascular senescence and atherosclerosis, which are recuperated by angiotensin II type 1 receptor (AT1R) signal inhibition. Mitochondria are dynamic organelles that undergo fusion and fission. This study aims to clarify whether mitochondrial dynamics is involved in the effects of metabolic stress on metabolic stress-induced vascular senescence. We used C57BL6 (WT), apolipoprotein E (ApoE) deficient and the double knockout of ApoE and AT1R (DKO) mice in in vivo study. We also performed in vitro experiment using human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC). Results: The degree of vascular senescence were higher in ApoE than those in WT. The number of fused mitochondria and mitochondrial function measured by ATP production were lower and mitochondrial oxidative stress assessed by Mito-sox Red staining and the number of apoptotic cell were higher in ApoE KO than those of WT. Treatment with mdivi-1 which is a specific inhibitor of mitochondrial fission factor, Drp-1, to ApoE KO reduced mitochondrial fission, oxidative stress and apoptosis, and attenuated vascular senescence. Administration of either ox-LDL or AGE to either HUVEC or VSMC induced mitochondrial fission, its dysfunction and apoptosis, which were restored by mdivi-1. These results suggest that metabolic stress induces mitochondrial fission and its dysfunction resulting in cellular senescence. Inhibition of AT1R by AT1R blocker ameliorated excessive mitochondrial fission, its function and oxidative stress induced by either ox-LDL or AGE. We also demonstrated that the number of fused mitochondria and its function were higher and the degree of vascular senescence and the number of apoptotic cell were lower in DKO than those in ApoE KO mice. Taken together, these results suggest that either ox-LDL or AGE causes cellular/vascular senescence through AT1R signal-dependent mitochondrial fission and subsequent mitochondrial dysfunction. Mitochondrial fission and subsequent mitochondrial dysfunction induced by ox-LDL or AGE play a crucial role to develop cellular/vascular senescence through AT1R signal.

P04-8Angiotensin Receptor Neprilysin Inhibitor Preserved Cardiac Function in Atherosclerotic MouseYasunori Suematsu1),�Kohei�Tashiro1),�Takashi�Kuwano1),�Shin-ichiro�Miura1)

1）Fukuoka UniversityIntroduction: Angiotensin receptor neprilysin inhibitor (ARNI) increases natriuretic peptide and improves heart failure. We previously reported that ARNI has the effects of suppression of myocardial hypertrophy and fibrosis in addition to reduction of afterload by natriuretic effect. Natriuretic peptide induces brown thermogenic adipose tissue and reduces atherosclerosis. Therefore, we investigated the effect of ARNI against mouse model of atherosclerosis. Method: Mouse model of atherosclerosis which apolipoprotein E knockout mouse fed a high cholesterol diet for 4 weeks was not treated (CTL) or treated with valsartan 30 mg/kg (VAL), sacubitril 30 mg/kg (SAC), or ARNI (VAL+SAC) 60mg/kg for 13 weeks. Blood pressure was measured by weekly and transthoracic cardiac echography was performed before treatment and after 13 weeks treatment. After treatment, aortic tissue was stained with Oil Red O for investigation of atherosclerosis. Results: Body weight was increased from 24.8±1.6 to 34.7±4.7 mg and each treated group did not show significant reduction of body weight compared to CTL group. Systolic blood pressure was increased from 114±12 to 123±13 mmHg and each treated group did not show significant reduction of body weight compared to CTL group. In baseline transthoracic cardiac echography, the parameters did not show significant differences between the groups. After 13 weeks treatment, left ventricular ejection fraction (LVEF) in CTL group was significantly reduced (81.6±4.8 % v.s. 67.8±8.2 %, p=0.001), but the other groups preserved LVEF after treatment than that in before treatment (VAL: 81.8±5.3 % v.s. 76.0±7.2 %, p=0.1, SAC: 79.4±3.7 % v.s. 76.1±7.5 %, p=0.3, ARNI: 78.9±5.3 % v.s. 76.6±6.7 %, p=0.5). After treatment, LVEF in only ARNI group was significantly preserved than that in CTL group (CTL: 67.8±8.2 % v.s. ARNI: 76.6±6.7 %, p<0.05). Although the rate of average atherosclerotic area was 16.3±5.8 %, there was no significant differences between the groups. Conclusion: Although ARNI did not suppress macro-atherosclerosis, it preserved LVEF. ARNI may suppress micro vascular injury in mouse model of atherosclerosis.

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P05-4The Potent p300-HAT Inhibitor GO-Y030 Suppresses TAC-induced Development of Heart Failure in miceKana Shimizu1,2),�Masafumi�Funamoto1,2),�Yoichi�Sunagawa1,2,3),�Yasufumi�Katanasaka1,2,3),�Yusuke�Miyazaki1,2,3),�Koji�Hasegawa1,2),�Tatsuya�Morimoto1,2,3)

1）Division of Molecular Medicine, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, 2）Clinical Research Institute, Kyoto Medical Center, National Hospital Organization, 3）Shizuoka General HospitalIntroduction: We previously found that curcumin, a natural p300 histone acetyltransferase (HAT) inhibitor, suppresses cardiomyocyte hypertrophy and the development of heart failure. However, curcumin has low bioavailability; therefore, it is important to identify curcumin analogues which possess strong therapeutic potential. In the present study, we focused on GO-Y030, which possesses stronger anti-cancer activity than curcumin, and investigated whether GO-Y030 is sufficiently potent for heart failure therapy. Methods Results: First, an in vitro p300 HAT assay revealed that GO-Y030 also possessed p300-HAT inhibitory activity, and that the IC50 value of GO-Y030 was 1.1 µM, while that of curcumin was 9.4 µM. Moreover, examination of the structure–activity relationship of GO-Y030 revealed that its 4 alkoxy groups (3, 3’, 5, 5’) were more important than its mono-ketone moiety for its p300-HAT inhibition activity. Second, cultured cardiomyocytes were treated with GO-Y030 or curcumin and then stimulated with phenylephrine (PE). 1 µM of GO-Y030 suppressed the following effects to the same extent as 10 µM of curcumin: PE-induced histone H3K9 acetylation, increases in the mRNA levels of ANF and BNP, and an increase in the surface area of cardiomyocytes. Third, C57BL/6J male mice were subjected to transverse aortic constriction (TAC) or sham operation. One day after the operation, the TAC mice were randomly assigned to five groups: vehicle, 1 or 50 mg/kg curcumin, and 0.1 or 0.5 mg/kg GO-Y030. Oral administrations were repeated for 6 weeks. Echocardiographic analysis showed that 0.5 mg/kg GO-Y030 prevented a TAC-induced increase in posterior wall thickness and systolic dysfunction to the same extent as 50 mg/kg curcumin. Moreover, 0.5 mg/kg GO-Y030 suppressed increases in the following parameters to the same extent as 50 mg/kg curcumin: HW/BW ratio, myocardial cell diameter, perivascular fibrosis, mRNA levels of ANF and BNP, and histone H3K9 acetylation. Conclusions: These results indicate that the curcumin analog GO-Y030 inhibits p300-HAT activity more strongly than curcumin and its derivatives in vitro, and that a low dose of GO-Y030 can prevent both cardiomyocyte hypertrophy and the development of heart failure in mice. These findings suggest that GO-Y030 may be an effective agent for heart failure therapy.

P05-1Protein kinase N promotes cardiac hypertrophy and fibrosisTeruhiro Sakaguchi1), � Mikito� Takefuj i1), � Toyoaki�Murohara1)

1）Nagoya university

Background—Heart failure, a common and potentially fatal cardiovascular disease, is caused by cardiac remodeling in response to pressure or volume overload. Protein phosphorylation is an important regulatory mechanism mediates various cellular signal transduction in cardiac function. The RHOA-associated protein kinase (ROCK/Rho-kinase) is known as an effector controled by the small GTPase RHOA. ROCK/Rho-kinase causes pathological phosphorylation of proteins and plays an important role in cardiac dysfunction. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiac remodeling is not understood. Methods—To investigate the role of PKNs in cardiac dysfunction, we generated mice with cardiomyocyte-specific deficiency for PKN1 and PKN2, and we performed transverse aorta constriction (TAC) surgeries and continuous angiotensin II (ANGII) infusion as a model of heart failure. Results—We showed that RHOA activates PKN1 and PKN2, the members of the PKN family of proteins, in cardiomyocytes of mice. In cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2-DKO) mice, no difference were observed. We found that cmc-PKN1/2-DKO mice were protected from TAC- and angiotensin II-induced hypertrophy and deterioration of LVFS. Plasma blood levels of brain natriuretic peptide (BNP) were significantly lower in cmc-PKN1/2-DKO mice. Moreover, we showed cmc-PKN1/2-DKO mice reduced the TAC- and angiotensin-induced increase of cardiomyocyte size and cardiac fibrosis.Conclusions—PKN1 and PKN2 play a role in regulating hypertrophy and fibrosis in the heart and can be promising therapeutic strategy for cardiac dysfunction.

P05-2Cavin-2/SDPR Deficiency Attenuates Myofibroblast Differentiation and Cardiac Fibrosis in TAC MiceYusuke Higuchi1),�Takehiro�Ogata2),�Naohiko�Nakanishi1),�Masahiro�Nishi1),�Akira�Sakamoto1),�Yumika�Tsuji1),�Satoaki�Matoba1)

1）Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, 2）Department of Pathology and Cell Regulation, Kyoto Prefectural University of MedicineIntroduction: Heart failure (HF) is a debilitating disease associated with high morbidity and mortality. A significant cause of HF is the adverse tissue remodeling with fibrosis. Excessive extracellular matrix (ECM) turnover is involved in the unfortunate outcome. Trans-differentiation of fibroblasts into myofibroblasts is a defining feature of fibrosis. Myofibroblasts express α-smooth muscle actin (αSMA) and secrete ECM proteins. The signals that control the trans-differentiation include the transforming growth factor (TGF)-β1-Smad pathway and Rho GTPase-dependent actin polymerization. Cavins and caveolins are the major protein components of caveolae. The previous report showed that some of cavin family proteins affect cardiac fibrosis and function. However, the structural role of Cavin-2/SDPR has been well elucidated, the role of Cavin-2/SDPR in cardiac fibrosis remains unknown.Objective: We investigated the functional role of Cavin-2/SDPR in cardiac fibrosis and cardiac function induced by pressure overload.Methods and Results: To examine the role of Cavin-2/SDPR in cardiac function, we performed transverse aortic constriction (TAC) operations on WT mice and Cavin-2-/- mice. Four weeks after TAC procedure, left ventricular fractional shortening was significantly preserved in Cavin-2-/- mice compared with WT mice. Also, the fibrosis area was significantly decreased in Cavin-2-/- mice. Moreover, mRNA expression of fibrosis-related genes was attenuated in the hearts of Cavin-2-/- mice four weeks after TAC. Immunohistostaining of mice hearts showed that αSMA positive cells were decreased in Cavin-2-/- mice after TAC. In vitro study, we isolated mouse embryonic fibroblasts (MEFs) from Cavin-2-/- mice and WT mice. Western blotting and immunostaining of MEFs showed that αSMA protein level was decreased in TGF-β1 stimulated MEFs derived from Cavin-2-/- mice. However, phosphorylation of Smad2/3 was not different in both WT MEFs, and SDPR-KO MEFs, SDPR deficiency significantly attenuated MRTF and SRF gene expressions in MEFs.Conclusions: Our observations suggest that Cavin-2/SDPR contributes to the development of cardiac fibrosis and systolic dysfunction with the differentiation of fibroblasts into myofibroblasts via the actin-MRTF-SRF gene transcription pathway.

P05-3Sirt7 plays a distinct role in cardiomyocyte and non-cardiomyocyte in response to pressure overloadSatoru Yamamura1),�Yasuhiro� Izumiya1),�Satoshi�Araki1),�Taishi�Nakamura1),�Toshifumi� Ishida1),�Yuichiro�Arima1),�Kenichi�Tsujita1)

1）Kumamoto University

Background: Sirt7, latest identified mammalian sirtuin, has been shown to modulate appropriate myocardial tissue repair after acute myocardial infarction. However, its role in the development of cardiac remodeling following chronic pressure overload is not clear. Methods and Results: Homozygous Sirt7-deficient (Sirt7-/-) and wild-type (WT) mice were subjected to pressure overload induced by angiotensin Ⅱ (Ang Ⅱ) infusion (2000 ng/mg/min) or transverse aortic constriction (TAC). In Ang Ⅱ infusion and TAC model, Sirt7 protein expression was increased in myocardial tissue after infusion and surgery. Ang Ⅱ -induced increase in heart weight/tibial length (HW/TL) was significantly attenuated in Sirt7-/- mice compared with WT mice. Interestingly, histological analysis showed that cross sectional area (CSA) was significantly larger, whereas fibrosis area was significantly smaller in Sirt7-/- mice. Consistent with these results, transcript expression of ANP andβMHC were significantly higher, whereas Collagen (col)- Ⅰ and Ⅲ were significantly lower in Sirt7-/- mice. In addition to these findings, Ang II-induced increase in HW/TL was significantly augmented in cardiomyocyte specific Sirt7-knockout (cKO) mice compared with control mice . Furthermore, histological analysis showed that CSA was significantly larger, and transcript expression of ANP and BNP were significantly higher in cKO mice. Cardiac contractile function was markedly decreased in cKO mice in this model. Consistent with these results, in vitro, phenylephrine induced cardiomyocyte hypertrophy was enhanced by Sirt7 knockdown.Conclusion: Sirt7 deficiency promotes cardiomyocyte hypertrophy, whereas suppresses interstitial fibrosis in response to pressure overload. The role of Sirt7 in cardiac remodeling suggested to be different in cardiomyocyte or non-cardiomyocyte.


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P05-5Amlexanox or Dantrolene prevents angiotensin II-induced cardiac hypertrophy in neonatal myocytesTetsuro Oda1),� Takuma�Nanno1),� Shigeki�Kobayashi1),�Takeshi�Yamamoto1),�Masafumi�Yano1)

1）Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of MedicineCardiac pathological hypertrophy occurs in response to pressure overload and myocardial infarction. The heart initially compensates for the overload by hypertrophic growth but function in the pathological hypertrophied heart eventually decompensates and, finally, leads to heart failure (HF). Thus, cardiac pathological hypertrophy is an important risk factor for cardiovascular disease. G protein-coupled receptor kinases, notably GRK5, which is most abundant in the heart, are crucial in regulating cardiac hypertrophy. GRK5 nuclear accumulation, which requires calmodulin (CaM) binding to GRK5, accelerated cardiac hypertrophy and HF. Thus, suppression of GRK5 nuclear accumulation by drug may be a novel therapeutic approach for preventing adverse pathological cardiac hypertrophy and HF. Methods and Results: First, we tested whether angiotensin II (AngII; which well known to stimulate pathologic hypertrophy) might cause cardiac hypertrophy in neonatal cardiac myocytes using immunofluorescence. AngII induced significant cardiac hypertrophy. To further test whether this hypertrophy related with GRK5 nuclear accumulation, we checked the GRK5 and CaM movements using amlexanox (AMX), which is an inhibitor of GRK5 kinase activity, and dantrolene, which is known to enhance CaM-RyR2 binding affinity. AngII induced GRK5 translocation to the nucleus together with CaM and treatment with AMX or dantrolene dramatically suppressed AngII-induced cardiac hypertrophy and prevented nuclear export of HDAC5, which promotes hypertrophic transcription.Conclusions: AngII-induced cardiac hypertrophy drives both GRK5 and CaM to the nucleus, but blocking GRK5 or CaM movement by AMX or by DAN prevents cardiac hypertrophy indicating that treatment with AMX or DAN maybe a novel potentially approach for preventing pathological cardiac hypertrophy.

P05-6A ryanodine receptor stabilizer as a novel strategy for pressure-overloaded heart failureMichiaki Kohno1)

1）Yamaguchi UniversityBackground: It remains unsolved how altered intracellular Ca2+ handling is associated with the transition to heart failure in pressure-overloaded mice.Aim: We investigated (1) whether transverse aortic constriction (TAC)-induced pressure overload caused Ca2+ leak through RyR2, (2) whether chronic administration of dantrolene, a RyR2 stabilizer, inhibited the transition to heart failure by stopping the Ca2+leak, and (3) the dantrolene treatment improves the prognosis in TAC mice.Methods: We developed heart failure model by 8 weeks-TAC in mice. TAC mice were divided into dantrolene-treated (DAN) group (dantrolene 20mg/kg/day, i.p.) and vehicle (non-DAN) group, and serial echocardiography was performed to compared cardiac function among DAN group, non-DAN group and sham-operated controls (Sham). Then, cardiomyocytes were isolated from hearts, and diastolic Ca2+ spark assay, simultaneous Ca2+ transient (CaT) and cell shortening (CS) assay were examined by confocal microscopy 2 weeks, 4weeks, and 8weeks after TAC. Survival rate was compared among 3 groups by Kaplan-Meier analysis. Results: Echocardiography showed that fractional shortening(FS) significantly decreased in non-DAN group, but it did not in DAN group at 4, 8 weeks-TAC, although there was no difference in FS between DAN and non-DAN groups. Similarly, %CaT and %CS were much higher in DAN group than in non-DAN group 4 and 8 weeks after TAC, although there was no difference between DAN and non-DAN groups. Interestingly, Ca2+ spark frequency (CaSF) was significantly higher in non-DAN group than in DAN group even at 2 weeks TAC. Kaplan-Meier survival analysis showed that DAN group significantly improved the prognosis as compared with non-DAN group. Conclusions: The aberrant Ca2+ leak through RyR2 was observed even in the compensated pressure-overloaded heart. Dantrolene prevented the transition to heart failure by inhibiting the aberrant Ca2+ leak through RyR2. Thus, a RyR2-stabilizer may be a novel strategy for pressure-overloaded heart failure.

P05-7Verification of novel molecular mechanism of cardiac hypertrophy via regulation of mRNA degradationYuki Masumura1),�Shuichiro�Higo1,2),�Shungo�Hikoso1),�Seiji�Takashima3),�Yasushi�Sakata1)

1）Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2）Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, 3）Department of Medical Biochemistry, Osaka University Graduate School of MedicineBackground Cardiac hypertrophy is initially an adaptive response to exogenous stimuli caused by either high blood pressure or neurohumoral factors, and is recognized as the causative mechanism during the progression of heart failure. We previously reported that Btg2, an immediate early gene which is transiently upregulated in cardiomyocytes under hypertrophic st imulat ion, repress cardiomyocyte hypertrophy via negatively regulating cytosolic RNA accumulation through interacting with CCR4-NOT deadenylation complex. The purpose of the study is to identify the target genes of Btg2 using high-throughput sequencing analysis, and to elucidate the novel mechanism of cardiomyocyte hypertrophy through regulation of RNA metabolism. Methods and Result To identify the genes targeted by Btg2 in cardiomyocyte under hypertrophic stimulation, we performed RNA sequence (RNA-seq) analysis using rat neonatal cardiomyocytes transfected with either two siRNA against Btg2 or non-targeted negative control siRNA and treated by phenylephrine, then we obtained dif ferential expression profiles in these cardiomyocytes. Recent biochemical studies clarify that Btg2 bridges CCR4-NOT complex and RNA binding protein and stimulates targeted mRNA degradation activity. To reveal the genes in cardiomyocytes bound by Btg2 via RNA binding protein under adrenergic stimulation, we performed RNA immunoprecipitation using specific antibody against Btg2 coupled with high throughput sequencing (RIP-seq). As a result, RNA-seq analysis identified 256 genes specifically upregulated in cardiomyocytes in which Btg2 was knocked down under adrenergic stimulation, and RIP-seq analysis identified 159 genes specifically bound by Btg2 via RNA binding protein under adrenergic stimulation. We identified 24 genes which were included in both sequencing data. Among these genes, known cardiac hypertrophy related genes were included. Conclusion RNA-seq analysis combined with RIP-seq analysis identified the subset of genes potentially involved in the regulation of cardiac hypertrophy via RNA degradation pathway.

P05-8A Novel Long Noncoding RNA Protects the Heart from Heart Failure Accompanied by Renal DysfunctionTakuya Kumazawa1), � Hitoshi� Nakagawa1), � Yasuki�Nakada1),� Tomoya�Nakano1),� Satomi� Ishihara1),� Kenji�Onoue1),�Yoshihiko�Saito1)

1）Cardiovascular Medicine, Nara Medical University, Japan

Patients with chronic kidney disease (CKD) are highly prone to cardiovascular diseases, including heart failure, cardiac hypertrophy, and atherosclerosis, named as the cardiorenal syndrome (CRS). However, the molecular mechanism underlying CRS remains unknown. Our previous study indicated that soluble Flt-1 (sFlt-1), an endogenous antagonist of placental growth factor (PlGF), can be a key molecule of CRS. Progression of renal disease decreases sFlt-1 production and its deficiency causes atherosclerosis progression. Herein, we identified a novel long noncoding RNA (lncX) associated with CRS. sFlt-1 deficient mice and their wild-type (WT) littermates were subjected to transverse aortic constriction (TAC), and gene expression was evaluated by microarray after 3 days. Novel lncRNA (lncX) was specifically reduced after TAC in hearts of sFlt-1 KO mice. The function of lncX was investigated by knockdown (KD) experiments using primary culture cells derived from mouse embryonic heart. KD of lncX induced proliferation and hypertrophy. Immunoblots showed that KD of lncX increased ERK1/2, but not MEK, phosphorylation. lncX localized in the cytoplasm and inhibited MAPK signaling by binding to MEK. To explore lncX function in vivo, we transduced mice with an adeno associated virus (AAV) vector encoding shRNA targeting non-specific control or lncX. KD of lncX caused heart failure by pressure-overload, as it aggravated cardiomegaly and lung to body weight ratios compared to that in WT mice after TAC. Echocardiography revealed that left ventricular (LV) wall thickening was significantly increased and LV ejection fraction was decreased; histological analysis showed that myocardial fibrosis was significantly increased in lncX KD compared to that in the control. Thus, lncX can be a novel therapeutic target for CRS with cardiac hypertrophy and heart failure.

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P06-4α1A-adrenergic receptor suppresses cardiac hypertrophy in pregnancy-associated hypertensive miceChulwon Kwon1),�Junji�Ishida2),�Akiyoshi�Fukamizu1,2)

1）Graduate school of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan, 2）Life Science Center of Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki, Japan

［Background］Hypertensive disorders of pregnancy (HDP) is one of the life-threatening diseases during pregnancy. The incidence of HDP is 8-10% of all the pregnancy in worldwide case. HDP patients show severe cardiovascular damages, such as hypertension and cardiac hypertrophy. Recently, development of HDP is suspected to relate to a vasopressor angiotensin II and its receptor (AT1). However, AT1-receptor blockers (ARBs) are prohibited because of their risk to induce teratogenicity. In recent years, adrenergic receptor blockers were approved to HDP patients for the purpose of lowering blood pressure, while the precise roles of adrenergic receptors in the pathological conditions of HDP are not well understood. Adrenergic receptors (ARs) consist of many subtypes. Recently, we found that α1A-subtype functionally interacts with apelin-receptor (APJ), which has high homology to AT1 receptor and regulates vascular contraction (unpublished). Interestingly, it was reported that the autoantibody against α1-subtypes including α1A-AR was abundant in the plasma of HDP patients (Guiling et al., 2013), implying the participation of α1A-AR in the progression of HDP. In this study, we examined the effect of α1A-AR deficiency on maternal and fetal abnormalities in pregnancy-associated hypertension using a mouse model of HDP. [Methods] We previously generated the pregnancy-associated hypertensive (PAH) mice, that exhibit similar phenotypes in HDP (Takimoto et al., 1996). To elucidate the functional importance of α1A-AR in PAH pathologies, we established PAH/α1A-AR-KO mice by using CRISPR/Cas9 system. Heart rates and systolic blood pressure were measured by tail-cuff methods during pregnancy. At the day before delivery, maternal organs and fetuses were harvested for detailed analyses. [Results] In PAH/α1A-AR-KO mice, heart rates were increased during late pregnancy, while maternal blood pressure was slightly but significantly attenuated. In addition, the lack of α1A-AR induced cardiac hypertrophy more severe than that in PAH mice. On the other hand, the reduction of maternal body weight gain and fetal intrauterine growth retardation observed in PAH mice were not recovered by α1A-AR deficiency. These results indicated that α1A-AR has the cardioprotective role on the pathological condition of PAH heart.

P06-1Analysis of molecular dynamics of TN-C in Kawasaki disease using a murine model induced by FK565Daiki Yamamoto1),�Yui�Toyofuku1),�Daisuke�Katoh1),�Zenjiro�Sakamoto1,2),�Hisanori�Nishio2),�Kyoko�Imanaka-Yoshida1)

1）Departmtent of Pathology and Matrix Biology, Mie University Graduate School of Medicine, 2）Department of Pediatrics, Graduate School of Medical Sciences, Kyushu UniversityKawasaki disease (KD) is an acute febrile illness of childhood characterized by systemic vasculitis and hyperendemic in Japan. The most critical complication is coronary aneurysm. High-dose intravenous immunoglobulin (IVIG) therapy has reduced the occurrence of coronary aneurysm, however, approximately 20 % of the patients show resistance to IVIG therapy. Our group has recently reported that high serum level of tenascin-C (TNC) KD patients have high risk of coronary aneurysm formation. To elucidate the role of TNC in pathophysiology of KD, we employed a mouse KD model induced by FK565, a nucleotide-binding oligomerization domain (Nod)-1 ligands. We injected 200μg / mouse of FK565 on day 1 and 5 into TNC reporter mice in which the LacZ gene was knocked in to one of the TNC location. We sacrificed the mice on day 7,14,22,45, and the hearts were immuno-histologically analyzed. At day 7, histological section showed severe inflammatory cell infiltration in coronary and aortic vascular walls and peri-adventitial areas, associated with destruction of elastic fibers, which were similar to the changes observed in KD patients. Inflammatory cell infiltration the gradually decreased, inflammation was subsided and mature collagen fibers were observed around the vessels and on day 45. Deposition of TNC was prominent at day 7 in the vascular lesion, gradually decreased and disappeared at day 45. Serum levels of TNC of the model mice measured by ELISA peaked at day7 and decreased at day 22, which corresponded to and local deposition of TNC in the vascular lesion and reflected the histological change. To identify the TNC producing cell, we stained the β-gal combined with immunostaining for various cell markers. We found that two type of cells produced TNC: vascular smooth muscle cells in the medial layer and mesenchymal cells in the adventitia positive for fibroblast marker, DDR2 and/or FSPl. Inflammatory cells did not synthesize TNC. In vitro, Il-1β and TNF-α upregulated the expression of TNC in human coronary smooth muscle cells. These results suggest that elevated serum TNC could reflect the reactivity of vascular smooth muscle cells and mesenchymal cells during inflammation and tissue repair/regeneration process in the vascular injury of KD.

P06-2Cocoa Bean Polyphenols Inhibit Pressure Overload-Induced Cardiac Hypertrophy and Heart FailureNurmila Sari1),�Yasufumi�Katanasaka1,2,3),�Hiroki�Honda1),�Yusuke�Miyazaki1,2,3),�Yoichi�Sunagawa1,2,3),�Koji�Hasegawa2),�Tatsuya�Morimoto1,2,3)

1）Division of Molecular Medicine, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 2）Division of Translational Research, Kyoto Medical Center, National Hospital Organization, 3）Shizuoka General HospitalCardiovascular disease (CVD) is one of the leading cause of death in Japan. Pathological stress such as pressure overload and myocardial infarction stimulates cardiac hypertrophy, which increases the risk of heart failure (HF). Polyphenols are natural compounds that extensively used for the prevention and treatment of CVD. Cocoa bean contains a high amount of polyphenols that have been consumed for centuries not only because of delighted taste but also its beneficial effect. However, the effect of cocoa bean polyphenols (CBPs) on the development of cardiac hypertrophy and HF is not apparent. The purpose of this study is to examine the effect of CBPs on pressure overload-induced cardiac hypertrophy and HF.Primary cultured cardiomyocytes from neonatal rats were treated with CBPs and then stimulated with phenylephrine. The cell surface area, hypertrophic gene transcriptions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), and phosphorylation of ERK1/2 and GATA4 were measured in the cardiomyocytes. C57BL/6j male mice were subjected to sham or transverse aortic constriction (TAC) operation. The mice were orally administrated with vehicle or CBP for eight weeks as follows: sham-vehicle, TAC-vehicle, TAC + CBP 600 mg/kg/day (CBP-L), or TAC + CBP 1200 mg/kg/day (CBP-H), and cardiac hypertrophy and function were examined.CBPs significantly suppressed PE-induced cardiomyocyte hypertrophy, hypertrophic gene transcriptions, and ERK1/2 and GATA4 phosphorylation. Echocardiography showed that fractional shortening (FS) was reduced in TAC-operated mice. The cardiac dysfunction was improved by treatment with CBPs at 1200 mg/kg (39.9% vs 47.9%, p<0.05). The CBPs treatment significantly reduced left ventricular posterior wall end of diastole and heart weight/tibia length ratio (HW/TL) compare with TAC + vehicle group (7.43 mg/mm vs 9.26 mg/mm, p<0.05). Myocardial cell hypertrophy, cardiac fibrosis and hypertrophic gene transcriptions induced by pressure overload were significantly suppressed by the treatment with CBPs. CBP treatment inhibited pressure overload-induced ERK1/2 phosphorylation in hearts.Conclusion: CBPs significantly suppressed pressure overload-induced cardiac hypertrophy and systolic dysfunction through ERK1/2/GATA4 pathway, at least in part, in cardiomyocytes.

P06-3Guanylyl Cyclase-A Signaling Attenuates the Deleterious Salt Effect on Mineralocorticoid ReceptorHitosh i Nakagawa 1), � Takuya� Kumazawa 1), � Yuko�Yoshioka1),� Tomoko� Ioka1),� Tomoya�Nakano1),� Kenji�Onoue1),�Yoshihiko�Saito1)

1）Nara Medical University

Background: Sodium causes the development of cardiovascular disease such as hypertension, cardiac hypertrophy and heart failure in conjunction with enhanced renin-angiotensin-aldosterone system (RAAS). Natriuretic peptide (NP), which is an important sodium regulator , prevents pathological cardiac alternations by counteracting RAAS. However, it is not elucidated whether NP inhibits sodium-effect on adverse cardiac alternations. We investigated whether salt excess exacerbates cardiac remodeling in mice with impaired NP signaling.Methods and Results: Mice lacking the gene encoding the NP receptor (guanylyl cyclase (GC)-A) and wild type (WT) mice were assigned to vehicle or subpressor dose of aldosterone (100 ng/kg/min) administration group under low salt (0.001% NaCl), normal salt (0.6% NaCl) and high salt diet (6.0% NaCl) for 4 weeks. Salt load did not induce cardiac change in both vehicle and aldosterone groups in WT mice. On the other hand, cardiac hypertrophy and interstitial fibrosis were significantly exacerbated in a salt dependent manner in aldosterone groups of GC-A KO mice, associated with enhanced gene expression relevant to hypertrophy, fibrosis and oxidative stress (BNP, collagen1 and Nox4, respectively). Of note, salt excess increased the expression of Sgk1, an important downstream of mineralocorticoid receptor (MR), in aldosterone groups of GC-A KO mice. These molecular changes were not observed in WT mice. Conclusion: The present study demonstrates that salt excess induces cardiac remodeling in conjunction with aldosterone in GC-A KO, but not in WT mice. These data indicate that the GC-A signaling attenuates the deleterious salt effect on aldosterone-induced cardiac remodeling.


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P06-5Left Ventricular Global Longitudinal Strain Values in Healthy Filipino Adults: Cross sectional StudyJulius Caesar de Vera1)

1）St. Luke's Medical Center-Global City

Background: Speckle tracking analysis of the left ventricle has only been recently available in the Philippines. This study was done to determine the mean normal values for global longitudinal strain of the left ventricle in normal healthy adult Filipinos.Methods:This was a retrospective, cross sectional study of normal, healthy adult Filipinos.Results: Two-dimensional speckle tracking analysis was done in 142 normal healthy adult Filipinos. Mean age was 39 + 9 years. The mean global longitudinal strain was -22.1 + 2.3%. Females had higher strain values compared to males, (-23.1 + 2.2% vs -21.1 + 1.8%, p value =<0.001). Normal BMI subjects had higher strain values as well compared to those with overweight BMI, (-22.5 + 2.3% vs -21.2 + 2.0%, p value = 0.001). Strain values by age group had no significant difference.Conclusion: This study was able to determine the mean global longitudinal strain of normal healthy adult Filipinos.

P06-6Right Ventricular Strain in Healthy Adult Filipinos A Retrospective Cross Sectional Pilot StudyJulius Caesar de Vera1),�Jonnah�Fatima�Pelat1)

1）St. Luke's Medical Center-Global City

Background: Speckle-tracking analysis, which was originally used for the assessment of left ventricular systolic function, can also be used in the assessment of the right ventricular systolic function. This was the first study to determine right ventricular strain of normal healthy adult Filipinos. Methods: This was a retrospective, cross sectional study done on normal, healthy, adult Filipinos. Results: Two-dimensional speckle-tracking analysis of the right ventricular lateral free wall was done in 66 normal healthy adult Filipinos (53% were male) with mean age of 39 + 9 years. The mean right ventricular lateral free wall strain was -24.0 + 4.5%. No correlation was seen with TAPSE and Sm’. However, with regards to the different right ventricular lateral free wall segments, the apical lateral free wall segment was shown to have significant association with TAPSE.Conclusion: This study was able to determine the mean normal right ventricular lateral free wall strain among normal healthy adult Filipinos.

P06-7Change of exhaled acetone concentration levels in patients with acute decompensated heart failureTetsuro Yokokawa1),� Akiomi� Yoshihisa1),� Takafumi�Ishida1),�Yasuchika�Takeishi1)

1）Department of Cardiovascular Medicine, Fukushima Medical University

Background: Although breath analysis has emerged as a noninvasive tool in several clinical conditions, it is not widely used in cardiovascular disease yet. Exhaled acetone is one of the compounds expected as biomarkers for heart failure. However, it is unknown how exhaled acetone concentration changes in clinical course of heart failure.Objective: To investigate time course of exhaled acetone concentration in acute decompensated heart failure.Methods: This study included 19 patients with acute decompensated heart failure (ADHF group), and 14 stable patients (control group). Exhaled acetone was collected from these patients and the concentration was measured with gas chromatography.Results: The ADHF group had higher heart rates (p=0.020), higher levels of brain natriuretic peptide (p < 0.001), and blood total ketone bodies (p=0.003), compared with the control group. In ADHF group, exhaled acetone concentration was significantly decreased after treatment (median: 2.40 ppm vs. 0.92 ppm, p < 0.001). On the other hand, in the control group, exhaled acetone concentration did not significantly change (median: 0.69 ppm vs. 0.62 ppm, p=0.370). Conclusions: Exhaled acetone concentration in patients with acute decompensated heart failure was drastically decreased by treatment, and therefore, could be a novel noninvasive biomarker to evaluate the course of acute decompensated heart failure.

P07-1Transcriptional regulation and physiological significance of ALK1 target genes in endothelial cellsNorika�Liu 1),�Yukihiro�Harada1,2),�Toru�Tanaka1),�Teruhisa�Kawamura2),�Yoshihiko�Saito3),�Yusuke�Watanabe1),�Osamu Nakagawa1)

1）Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, 2）Department of Biomedical Sciences, Ritsumeikan University College of Life Sciences, 3）The First Department of Internal Medicine, Nara Medical UniversityBone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases such as hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension, and the Alk1/Acvrl1 deletion in mice causes severe defects of vascular formation and embryonic lethality.　ALK1 signaling controls endothelial functions through transcriptional regulation of downstream target genes such as ID1/-2, SMAD6/-7, BMPR2 and HEY1/-2. We recently identified SGK1 and TMEM100 as novel downstream targets of ALK1 signaling. Expression of SGK1, serum/glucocorticoid-regulated kinase 1, in cultured endothelial cells is rapidly up-regulated by the BMP9 treatment through SMAD-mediated transcriptional activation. The Sgk1 expression in mouse embryos is enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice show embryonic lethality due to abnormal vascular formation, and its mRNA and protein levels are clearly reduced in Alk1/Acvrl1 null embryos. On the other hand, TMEM100 encodes a small transmembrane protein of as yet incompletely characterized functions, while Tmem100 null mice die in utero due to impairment of endothelial differentiation and vascular morphogenesis. TMEM100 expression is markedly but gradually increased by ALK1 signal activation in cultured endothelial cells, which likely requires production of unspecified signaling protein(s) in addition to or downstream of SMAD-dependent transcription. An enhancer conserved in mice and humans is capable of driving reporter expression specifically in endothelial cells of large caliber arteries, which reproduces the Tmem100 expression pattern in mouse embryos. We are further studying regulatory mechanisms of Sgk1 and Tmem100 expression, especially how ALK1 signaling cooperates with endothelial transcriptional programs, as well as their mode of actions in embryonic vasculature. Identification of novel target genes helps understand how ALK1 signaling is involved in normal vascular development and etiologies of human diseases.

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P07-5tie1 but not tie2 is essential for cardiovascular development in zebrafishNanami Morooka1),�Hiroyuki�Nakajima1),�Ayano�Chiba1),�Naoki�Mochizuki1)

1）National Cerebral and Cardiovascular Center

Angiopoietin − Tie signaling is essential for cardiovascular development in mammals. Deletion of Angiopoietin1 or Tie2 in mice results in impaired heart trabeculation and vascular network formation at E9.5. Angiopoietin2 and Tie1 mutants, on the other hand, show defects in lymphatic development at perinatal stage. Despite the importance of Ang − Tie signaling in mammals, tie2 mutant in zebrafish show no cardiovascular defect. To investigate whether the ang − tie signaling is essential for zebrafish development, we generated mutant fish of another tie receptor tie1 and angiopoietins (angpt1, angpt2a, and angpt2b) using transcription activator-like effector nucleases (TALEN). Whereas heterozygous mutant of tie1 was viable and fertile, homozygous mutant showed severe edema around gut, heart, and eyes at 5 dpf. We found that the thoracic duct, zebrafish trunk lymphatic vessel, was disrupted in tie1 homozygous mutant at 5 dpf. Lymphatic endothelial cells originate from venous endothelial cells of posterior cardinal vein (PCV). At around 36 hpf, secondary angiogenic sprouts come from PCV and become either intersegmental vein or parachordal lymphangioblasts (PLs) in the wild-type embryos. In tie1 homozygous mutant, secondary sprouts did not occur and failed to form PLs. The seconday sprouts of angpt1, angpt2a, angpt2b, and angpt2a/2b double homozygous mutant were normal. Even though the head vasculature in tie1 homozygous mutant was once formed at 2 dpf, it was regressed at 3 dpf. This regression is found in the embryos without blood flow in the brain. The tie1 homozygous mutant showed no flow in the heart at 3dpf. Therefore, tie1 is essential for zebrafish cardiovascular development.

P07-2Signif icance of Hey transcriptional factors in endothelial cells during cardiovascular developmentYusuke�Watanabe1),�Tomoe�Nakamura-�Nishitani1),�Daiki�Seya1),�Dai�Ihara1,2),�Teruhisa�Kawamura2),�Osamu Nakagawa1)

1）Department of Molecular Physiology, National Cerebral and Cardiovascular Center Research Institute, 2）Department of Biomedical Sciences, Ritsumeikan University College of Life Sciences

We and others previously identified the Hey family of bHLH transcription factors as mediators of Notch signaling in the developing cardiovascular system. Among three family genes, the expression of Hey1 and Hey2 was markedly enhanced by the activation of ALK1 signaling in cultured endothelial cells (EC) and was significantly down-regulated in EC of Alk1/Acvrl1 null embryos, suggesting that Hey1 and Hey2 worked also downstream of bone morphogenetic protein-ALK1 signaling. Embryos null for Hey1 or Hey2 did not show early defects of vascular formation, but combined loss of Hey1 and Hey2 caused embryonic lethality due to vascular demise similar to that observed in Notch and ALK1 signal deficient embryos. EC-specific deletion of Hey1 and Hey2 led to abnormal vascular morphogenesis virtually identical to the global double knockout phenotypes, indicating that shared functions of Hey1 and Hey2 in EC were indispensable for early vascular development. Additionally, Hey1 null mice showed specific defects of the fourth pharyngeal arch artery at late embryonic stages, resulting in great vessel anomalies such as the interruption of the aortic arch and perinatal lethality. Such defects were in a sharp contrast to the Hey2 null phenotypes, ventricular septal defect, tricuspid atresia and right ventricle hypoplasia, in cardiac morphogenesis. Great vessel anomalies were caused also by the EC-specific Hey1 deletion, indicating that endothelial Hey1 played a specific role in pharyngeal arch artery formation. We further demonstrated that embryonic expression of Hey1 in the vasculature and other tissues was maintained by a combination of tissue-specific enhancers. Thus, the members of the Hey transcription factor family, especially Hey1 and Hey2, play unique as well as overlapping roles in EC, which are essential for early and late vascular development.

P07-3A novel origin of ECs; Isl1-positve cells contribute to caudal vessel formation in zebrafishHiroyuki Ishikawa1),�Hiroyuki�Nakajima1),�Hajime�Fukui1),�Naoki�Mochizuki1)

1）National Cerebral and Cardiovascular Center Research Institute

Endothelial cells (ECs) are generally considered to originate from the lateral plate mesoderm (LPM). LPM-derived multipotent mesodermal progenitors differentiate into ECs and hematopoietic cells in a manner dependent on Cloche/Npas4l, a master transcription factor. However, even in the zebrafish cloche mutant, ECs are still present in the tail, while those are absent in the trunk region, suggesting Cloche-independent differentiation of ECs or other source of ECs rather than the LPM. We here report a novel origin of ECs in the tail of zebrafish. During somitogenesis, the body length must be extended. Following the extension of body, blood vessels also extend in the tail region. For the extension of blood vessels, there are two possibilities. One is that ECs proliferate and migrate toward the tail from the trunk. The other is that new ECs emerge in the tail region. Previously, we confirmed the contribution of proliferating EC to elongation of blood vessels in the tail. In this study, we found that Islet1 (isl1)-expressing cells give rise to ECs in zebrafish tail vasculature. Isl1 is well known as a marker of the second heart field (SHF). In BAC transgenic zebrafish line that expressed GFP under the isl1 promotor, GFP was expressed in the ECs of posterior blood vessels as well as SHF cells. In another BAC transgenic line that expressed destabilized EGFP (dEGFP, T1/2~2h) under the isl1 promotor, dEGFP-expressing cells became fli1 promoter-activated ECs in the tail region. The intensity of dEGFP became diminished as the cells differentiated into fli1 promoter-activated ECs, suggesting that isl1 is transiently expressed in the cells that differentiate into ECs. Thus, our data strongly suggest that Isl1-positive cells serve as a novel origin of endothelial cells in zebrafish tail vasculature.

P07-4Identification of new myocardial growth factor and its application to cardiac regenerationSho Haginiwa1),�Hiroyuki�Yamakawa1),�Naoto�Muraoka1),�Taketaro�Sadahiro1),�Hidenori�Kojima1),�Hidenori�Tani1),�Masaki�Ieda2)�1）Keio University, 2）Tsukuba University

Cardiomyocytes divide in the fetal stage. However, it is known that the cell division stops after birth. Therefore, once mature heart is suffered with myocardial infarction, the cardiomyocytes are not increasing in number and the contractile function is lost. In this study, we introduced genes into cardiomyocytes in the stage of contractile mal-function. As a result, the ability of dividing was reacquired cardiomyocytes increased and proper contractile function of the heart was recovered.First, we incorporated several genes that are specifically expressed in the fetal stage into lentiviral vectors are introduced into the cardiomyocytes of newborn rat in vitro, and the expression of cell cycle markers was analyzed by flow cytometry. As a result, gene X was identified as a specifically increased cell cycle marker expression in cardiomyocytes Next, an adeno-associated viral vector expressing the gene X was prepared and introduced into newborn mouse in vivo. Expression of the gene X was identified in immature cardiomyocytes in newborn mouse, and an increase of cell cycle marker expression was confirmed. And the gene X was introduced into adult mouse with an adeno-associated-vector to analyze the expression of cell cycle markers. As a result, we confirmed the cell cycle marker expression was increased in the cardiomyocytes of the adult mouse.Finally, we analyzed the effect of gene X for the gene expression of cardiomyocytes. Using the RNA which extracted newborn and adult mouse’s whole hearts, we analyzed by microarray. As a result, we conformed the gene expression of cell cycle is up-regulated and gene X affected the only cardiomyocytes


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P07-6Myocardial canonical Wnt signaling in zebrafish heart developmentAyano Chiba1),�Naoki�Mochizuki1)

1）National Cerebral and Cardiovascular Center Research Institute

The canonical Wnt signaling pathway is spatiotemporally activated during cardiac development, resulting in heart tube looping and atrioventricular valve formation. It is also linked to cardiac diseases such as myocardial infarction and arrhythmogenic cardiomyopathy. However, it remains unclear when and where the signaling is activated in cardiomyocytes (CMs) during cardiac development. To elucidate our question, we aimed to visualize beta-catenin (Ctnnb)-dependent transcriptional activation in CMs. Using GAL4/UAS system, we established a transgenic (Tg) zebrafish line that expressed fluorescent protein responding to Wnt/Ctnnb signaling activation in CMs (Ctnnb-reporter). The reporter embryo showed that Ctnnb signaling was activated exclusively at the atrioventricular canal (AVC) region. Previous papers reported that myocardial Ctnnb signaling induced Bmp2/4 expression in CMs and subsequently activated Tbx2-mediated AVC induction and valve formation. To simultaneously visualize spatiotemporal Ctnnb and Bmp activation during cardiac development, we crossed Ctnnb-reporter with Bmp signaling reporter Tg line. Surprisingly, myocardial Bmp signaling was activated in the ventricular side of AVC in contrast to Ctnnb signaling-activated CMs in the atrial side. We also confirmed that Ctnnb signaling-activated CMs did not overlap with Tbx2b expressing CMs. These spatial difference of signaling activation pattern suggests that CMs at the AVC can divide into several regions by the pattern of signal activation. Now we are trying to deplete Ctnnb signaling-activated CMs by nitroreductase-mediated cell ablation method for understanding their functions in AVC. In conclusion, we here newly identified canonical Wnt/Ctnnb signaling-activated CMs and reported potential roles of those cells in zebrafish cardiac development.

P07-7Extended in vitro culture enhanced maturation and engraftment of human iPSC-derived cardiomyocytesShin Kadota1,2),�Yuki�Tanaka1,3),� Satomi�Okano1,2),�Yuko�Wada3),�Kenji�Okada3),�Koichiro�Kuwahara4),�Yuji�Shiba1,2)

1）Department of Regenerative Science and Medicine, Shinshu University, 2）Institute for Biomedical Sciences, Shinshu University, 3）Department of Cardiovascular Surgery, Shinshu University, 4）Department of Cardiovascular Medicine, Shinshu UniversityBackground: Cell immaturity is one of the remaining hurdles that restrict the use of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) in drug development, disease modeling, and regenerative medicine. Although long-term in vitro culture and the in vivo environment are known to enhance the maturation of PSC-CMs, no studies have compared the development of PSC-CMs in these two conditions. Methods: Human iPSCs (253G1) were transdifferentiated into cardiomyocytes with a monolayer culture using Activin A, BMP4, and Wnt modulators. Cells cryopreserved at 28 and 56 days after differentiation were termed 4w PSC-CMs and 8w PSC-CMs, respectively. We transplanted 2 x 107 PSC-CMs into athymic rat hearts 7 days after the induction of myocardial infarction. To compare the maturity and graft size at equivalent ages of PSC-CMs, we histologically assessed rat hearts transplanted with 4w PSC-CMs on days 56 and 84 post-transplantation (n=5, each) and those with 8w PSC-CMs on days 28 and 56 (n=4, each). Results: Sarcomere length, cardiac troponin I, and other maturation markers were significantly enhanced in 4w PSC-CM grafts on day 84 compared with those on day 56, suggesting in vivo maturation of PSC-CMs. The expression of cardiac troponin I in 8w PSC-CM grafts on day 28 was significantly higher than that in 4w PSC-CM grafts on day 56. Interestingly, the graft sizes of 8w PSC-CMs on day 28 and 56 (mean: 6.3 mm2, n=8) were significantly larger than those of 4w PSC-CMs on day 56 and 84 (mean: 3.0 mm2, n=10) post-transplantation (unpaired t-test, P=0.037). Conclusion: Although PSC-CMs matured in a time-dependent manner in vivo, an extended in vitro culture period appeared to more remarkably enhance maturation and engraftment of PSC-CMs after transplantation.

P07-8EFFICIENT CARDIAC REGENERATION THERAPY BY CELL CYCLE ACTIVATION OF IPSC-DERIVED CARDIOMYOCYTESMnabu Kasamoto1),� Shunsuke� Funakoshi2),� Takeshi�Hatani2),�Takeshi�Kimura1),�Yoshinori�Yoshida2)

1）Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 2）Dept. of Life Science Frontiers, Center for iPS Research and Application, Kyoto UniversityCardiac transplantation therapy using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a promising strategy to cure very severe heart failure. We have previously reported that engrafted iPSC-CMs proliferate for three months after direct injection of the cells into the myocardium of immunodeficiency mouse infarcted hearts with left anterior descending artery (LAD) ligation. We also confirmed the iPSC-CMs is more proliferative in vivo than in vitro. Here, we analyzed the cell cycle of iPSC-CMs with the aim of augmenting engraftment efficiency after transplantation. We established an iPSC line constitutively expressing Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), which let us observe the cell cycle phase: Cells in a state of S/G2/M phase (proliferating stage) emit green fluorescence, cells in a state of G0/G1 phase (non-proliferating stage) red fluorescence. Next, we compared the engraftment ability between S/G2/M phase iPSC-CMs and G0/G1 phase iPSC-CMs, three months after transplantation. Cell cycle activated (S/G2/M phase) iPSC-CMs showed significantly higher engraftment efficiency than those in inactivated (G0/G1 phase). High-throughput screening of drugs for their ability to activate the cell cycle in iPSC-CMs was performed to realize effective engraftment. Out of 4032 initially screened compounds, 82 candidates emerged. Second screening identified some group of promising compounds. Among those, we focused on the most effective compound (CCA-1) and optimized the condition of the compound administration. Finally, we achieved the ideally increase in iPSC-CMs in S/G2/M phase from 2.8% to 39.8% via flow cytometry analysis. Next, we checked the proliferative effect of CCA-1 in other ways: EdU & cell number count.EdU assay showed CCA-1 enhanced DNA replication in those iPSC-CMs implying an increased proliferation ability. Similar to the above results, CCA-1 also increased cell number count. In vivo, CCA-1 treated iPSC-CMs showed enlargement of graft size after transplantation into mice ischemic heart. Altogether, these data demonstrate that cell cycle plays a key role in the effectiveness of cardiac cell therapy using iPSC-CMs, and its modification might lead to novel therapeutic treatments.

P08-1Development of PCSK9 epitope vaccine for dyslipidemia in miceHironori Nakagami1)

1）Osaka University

In our group, we try to develop the novel therapeutic vaccine for life-stule-related diseases, which mainly induces antibody production without the activation of cytotoxic T-cells. In the treatment of dyslipidemia, proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates expression of low-density lipoprotein (LDL) receptors via receptor internalization and subsequent lysosomal degradation. Thus, an anti-PCSK9 antibody is recently used as an anti-hyperlipidemia drug. Here, we aimed to develop vaccine for a long-term treatment of dyslipidemia targeted to PCSK9 instead of antibody therapy. We designed a peptide vaccine for mouse PCSK-9, which consisted of short peptides conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. Vaccines were administered to mice with adjuvants and significantly elicited an antibody response against PCSK9. The PCSK9 vaccines were administered to mice three times in 2-week intervals, and antibody titers and lipoprotein levels were evaluated up to 24 weeks after the first immunization to determine the therapeutic effect. Anti-PCSK9 antibody titers reached peak levels 6 weeks after the first immunization, and theses titers were maintained for up to 24 weeks. Decreased plasma levels of total cholesterol were maintained for up to 24 weeks. Immunized mice exhibited a significant increase in cell-surface LDL receptor expression. Stimulation with KLH, but not PCSK9, induced the production of INF-gamma and interleukin-4 (IL-4), as determined with ELISPOT assays, thus indicating that PCSK9 vaccine did not elicit T-cell activation in our vaccine system. The present anti-PCSK9 vaccine induced long-lasting anti-PCSK9 antibody production and improved lipoprotein profiles. Thus, anti-PCSK9 vaccine could become a new option for the treatment of dyslipidemia as a long-acting therapy in future.

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P08-5The effect of mTORC1 inhibition on necroptotic signaling-mediated suppression of autophagic fluxKoki Abe1),�Toshiyuki�Yano1),�Takayuki�Miki1),�Masaya�Tanno1),�Atsushi�Kuno1,2),�Tatsuya�Sato1,3),�Tetsuji�Miura1)

1）Sapporo Medical University, Department of Cardiovascular, Renal and Metabolic Medicine, 2）Sapporo Medical University, Department of Pharmacology, 3）Sapporo Medical University, Department of Cellular Physiology and Signal TransductionBackground: Necroptosis, a form of programmed necrosis, and autophagy, a process with ambivalent roles in cell survival, have been suggested to be involved in heart failure and ischemia/reperfusion injury. Our recent study showed that rapamycin, an mTORC1 inhibitor, improved suppression of autophagy induced by TNF-α (TNF) receptor activation, leading to suppression of cardiomyocyte necroptosis.　Purpose: We examined the mechanism by which rapamycin suppresses necroptosis of cardiomyocytes, focusing on regulation of RIP1 activity and autophagic flux.　Methods and results: A necroptotic pathway was activated by TNF (50 ng/ml) alone or co-incubation of TNF with 20 µM z-VAD-fmk (zVAD), a pan-caspase inhibitor, in H9c2 cardiomyoblasts. Extent of cell death was determined as LDH release expressed as % of total cell LDH. TNF/zVAD increased LDH release from 3.4±1.3% to 46.1±2.3%, which was suppressed by necrostatin-1, a RIP1 inhibitor, and by RIP3 or MLKL knockdown, confirming existence of RIP1/RIP3/MLKL-dependent necroptosis in cardiomyocytes. TNF/zVAD-induced cell death was attenuated by rapamycin, an mTORC1 inhibitor, and by Ku-0063794 (Ku), an mTORC1/2 inhibitor, but not by PF-4708671 (PF), a p70S6K inhibitor. Levels of necroptosis regulatory proteins upstream of RIP1 and TNF-induced cleavage of RIP1 were not changed by rapamycin. The protective effect of rapamycin on necroptosis was preserved in the presence of inhibitors of TAK1, IKKα/β, and cIAP, which are known as endogenous necroptosis suppressors. However, rapamycin increased putative RIP1-Ser320 phosphorylation that negatively regulates RIP1 activity, leading to suppression of TNF/zVAD-induced RIP1-RIP3 binding. Ku, but not PF, mimicked the effect of rapamycin on putative RIP1-Ser320 phosphorylation. Monitoring of autophagic flux using RFP-GFP-LC3 plasmids revealed that suppression of autophagic flux by TNF/zVAD was restored by rapamycin and by necrostatin-1 but not by RIP3 knockdown. Bafilomycin A1, a suppressor of autophagic flux, but not ULK1 knockdown, reversed the protective effect of rapamycin on TNF/zVAD-induced cell death.　Conclusion: mTORC1 inhibition induces inhibitory phosphorylation of RIP1, leading to restoration of cytoprotective autophagy and suppression of cardiomyocyte necroptosis.

P08-2Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug TestingHirohiko Kohjitani1),� Shigeya�Koda2),�Yukiko�Himeno2),�Takeru�Makiyama1),�Takeshi�Kimura1),�Akinori�Noma2),�Akira�Amano2)

1）Department of Cardiovascular Medicine, Kyoto University Hospital, 2）Graduate School of Life Sciences, Ritsumeikan University

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (-CMs) are expected tool for preclinical evaluation of the safety and efficacy of compounds on the heart. But, hiPSC-CMs exhibit varying action potential (AP) morphologies, and even exhibit paradoxical reaction for a well-known drug, i.e. E-4031.PURPOSE: To establish cell-specific in-silico hiPSC-CM models that can reproduce variety of hiPSC-CMs, and predict reaction of drug testing.METHODS: We developed new hiPSC-CMs mathematical models, adopting experimental data of ion-channels, and considering the features of hiPSC-CMs. We also developed AP processing system including noise reduction and voltage shift using inverse calculation of IKr open probability. We recorded APs from 60 hiPSC-CMs, and recapitulated all AP morphologies in simulation model by newly developed algorithmic auto-fitting system. After that, Multi-current blocking test was performed.RESULTS: We successful ly recapitulated all 60 AP morphologies of hiPSC-CMs from multiple cell lines. After IKr, IK1, ICaL, CDI-factor(KL), Iha, INa, voltage-shift of INa, INCX was included into fitting parameters, we can improved accuracy of re-creation, in the error range of 1.2mV per each point. When KL and voltage-shift of INa was excluded from fitting parameters, accuracy of re-creation was worsened to ±13mV per each point.CONCLUSION: AP processing and simulational re-creation system suggested that varieties of APs of hiPSC-CMs are related to not only conductance of each ion channel but also kinetics of ICaL and INa.

P08-3Development of a new high-throughput screening assay for the discovery of novel heart failure drugsHikaru Moki1),�Yoichi�Sunagawa1,3,4),�Yasufumi�Katanasaka1,3,4),�Naohisa�Ogo2),� Akira�Asai2),� Koji� Hasegawa1,3),� Tatsuya�Morimoto1,3,4)

1）Division of Molecular Medicine, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan, 2）The center for drug discovery, Graduate School of Pharmaceutical Societies, University of Shizuoka, Shizuoka, Japan, 3）Division of Translational Research, Clinical Research Institute, Kyoto Medical Center, National Hospital Organization, Kyoto, Japan, 4）Shizuoka General Hospital, Shizuoka, JapanIntroduction: Heart failure is the lethal end-stage of almost all heart diseases, including myocardial infarction and hypertension. Although pharmacological therapies for heart failure such as ARB, ACE inhibitors, and β-blockers are well established, the mortality of patients with severe heart failure remains high. Recently, high-throughput screening (HTS) assays are increasingly used for drug discovery. Compared with conventional assays, these assays can reduce cost, time, and technical errors. In this study, we have developed an HTS system to identify anti-hypertrophic compounds for heart failure therapy based on fluorescence microscopy imaging using primary neonatal rat cardiomyocytes. Methods: Primary cultured neonatal rat cardiomyocytes were incubated in 96-well plates for 36 hours. Then the cells were treated with one of two anti-hypertrophic compounds (α1-antagonist prazosin or curcumin), or with dimethyl sulfoxide (DMSO) as a control. After 2 hours, they were stimulated with 30 μM phenylephrine (PE) for 48 hours. Then, the cells were fixed with 3.7% paraformaldehyde. Myofibrillar organization was specifically stained with anti-α-actinin antibody and nuclei were stained with Hoechst 33258. Fluorescence images of α-actinin positive cardiomyocytes were automatically obtained using a Cellomics™ ArrayScan ® high-content screening system (Thermo Scientific), and their surface area were measured by HCS Studio™ Cell Analysis Software (Thermo Scientific).Results: The screening system could accurately select and evaluate the area of the α-actinin-positive cardiomyocytes automatically. The system also automatically detected PE-induced hypertrophy, as well as the effects of the anti-hypertrophic compounds prazosin and curcumin, which significantly inhibited that hypertrophy.Conclusion: These results indicate that this high-throughput screening assay based on fluorescence can be used to effectively discover anti-hypertrophic compounds. In future studies, we will attempt to identify novel cellular regulators of cardiomyocyte hypertrophy and potent heart failure drugs from chemical libraries using this “phenotype-based” HTS assay.

P08-4A bacteria-derived ACE2-like enzyme suppresses cardiac remodeling and dysfunction in mice.Takafumi Minato1),�Satoru�Nirasawa2),�Teruki�Sato1),�Ryo�Ozawa1),� Saori�Takahashi5),�Hiroyuki�Watanabe1),�Keiji�Kuba1)

1）Akita University Graduate School of Medicine, 2）Biological Resources and Post-Harvest Division, Japan International Research Center for Agricultural Sciences, 3）Bioscience Education and Research Support Center, Akita University Graduate School of Medicine, 4）Laboratory of Regulation of Intractable Infectious Diseases, National Institute of Biomedical Innovation, Health and Nutrition, 5）Akita Research Institute of Food and Brewing

Rationale: Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of renin-angiotensin system and has the beneficial effects on the cardiovascular diseases. Objective: To examine whether a bacteria-derived carboxypeptidase (BD-X) has ACE2-like enzymatic activity in vitro and vivo.Methods and Results: We prepared recombinant BD-X protein using bacterial protein expression system. Recombinant BD-X protein catalyzed the conversion of angiotensin II to angiotensin 1-7 with the same potency as rhACE2 in vitro. Treatment with BD-X reduced plasma angiotensin II levels and suppressed angiotensin II-induced hypertension, cardiac hypertrophy and fibrosis in mice. Moreover, continuous infusion of BD-X inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Conclusion: BD-X is an ACE2-like carboxypeptidase, which is functional in vitro and in vivo. BD-X could be a novel therapeutics in cardiovascular diseases. These results suggest that the strategy to find molecules of convergent evolution might be effective for drug development, such as ‘generic’ protein preparation of functional enzymes.


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P08-6Silencing of microRNA-34a ameliorates aortic valve calcification via Notch1-Runx2 signalingTaku Toshima1),�Tetsu�Watanabe1),�Tetsuro�Shishido1),�Takuya� Miyamoto 1), � Takanor i � Ar imoto 1), � H i rok i�Takahashi1),�Masafumi�Watanabe1)

1）Yamagata University School of MedicineIntroduction: Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease and is increased with increasing elderly population. However, the effective drug therapy has not been established. MicroRNAs (miRs) are approximately 20 bp noncoding nucleotides which have a regulatory role in gene expression. The aim of this study was to investigate the role of miRs in development of CAVS.Methods: We measured expression of miRs which were reportedly involved in mineralization using human aortic valve tissue from patients who underwent aortic valve replacement due to CAVS or aortic regurgitation. In vitro assay of miRs was examined using porcine aortic valve interstitial cells (AVICs) after treatment with osteogenic induction medium. We investigated whether a specific miR-inhibitor can suppress aortic valve calcification in a wire injury CAVS mouse model which we previously reported.Results: MiR-23a, miR-34a, miR34c, miR-133a, miR-146a, and miR-155 were increased in valve tissues from CAVS compared with those from aortic regurgitation. We chose miR-34a among increased miRs in porcine AVICs with osteogenic medium treatment, which were consistent with those from CAVS. Mineral deposition evaluated by alkaline phosphatase and alizarin red staining was significantly higher in porcine AVICs transfected with miR-34a mimic compared with those with miR-control. Notch1 expression was decreased, and runt-related transcription factor 2 (Runx2) expression was increased in miR-34a mimic transfected AVICs compared with those with miR-control. In contrast, inhibition of miR-34a significantly attenuated these mineralization signals in AVICs compared with miR-control. RNA pull down assay revealed that miR-34a directly binds Notch1 mRNA 3’ untranslated lesion and regulates its expression. In wire injury CAVS mice experiments, LNA miR-34a inhibitor suppressed aortic velocity, mineral deposition of aortic valves, and cardiac hypertrophy after wire injury, which were involved in decreased Runx2 and increased Notch1 expressions. Conclusions: MiR-34a plays an important role in development of calcific aortic valve stenosis via Notch1-Runx2 signaling. Inhibition of miR-34a may be the therapeutic target for CAVS.

P08-7A Novel Target Molecule of Nobiletin possess a Therapeutic Potency for Heart Failure in MiceYoichi Sunagawa1,2,3),� Yusuke�Miyazaki1,2,3),� Yasufumi�Katanasaka1,2,3),�Toshiyuki�Kan1),�Hiromichi�Wada2),�Koji�Hasegawa1,2),�Tatsuya�Morimoto1,2,3)

1）School of Pharmaceutical Sciences, University of Shizuoka, 2）Clinical Research Institute, Kyoto Medical Center, 3）Shizuoka General HospitalIntroduction: Heart failure is associated with pathological growth of cardiomyocytes. To achieve effective oral pharmacological therapy for heart failure, we screened compounds isolated from natural products and found that nobiletin, derived from Citrus depressa, inhibited phenylephrine (PE)-induced hypertrophic responses in cardiomyocytes and prevented the development of heart failure in vivo. However, the target molecule of nobiletin in cardiomyocytes is unclear. In this study, we attempted to identify novel nobiletin-binding proteins by a proteomics approach and investigate its functional mechanism in mice.Methods and Results: Nobiletin-binding proteins were purified from rat heart using biotin-conjugated nobiletin or biotin alone and identified by LC-LC/MS-MS. One of them, nobiletin-binding protein 1 (NBP1) which related to cellular metabolic pathway, was identified as a novel target molecule of nobiletin. Pulldown assay demonstrated that nobiletin directly interacted with recombinant NBP1. In vitro enzyme assay showed that nobiletin enhanced NBP1 activity. Although NBP1 knockdown could not affect PE-induced hypertrophic response gene transcriptions, such as ANF and BNP, and cardiomyocyte hypertrophy, NBP1 knockdown failed to exhibit nobiletin-mediated anti-hypertrophic effects in cardiomyocytes. In contrast, overexpression of NBP1 significantly prevented PE-induced cardiomyocyte hypertrophy and gene transcriptions of ANF and BNP. NBP1-KO mice and WT mice were subjected to sham or transarotic constriction (TAC) and randomly divided into two groups: nobiletin (20 mg/kg/day) and vehicle. Oral administration was repeated for 8 weeks. Nobiletin significantly improved TAC-induced cardiac hypertrophy and systolic dysfunction in WT mice but not in NBP1-KO mice. Nobiletin also prevented TAC-induced increases in HW/BW rate, myocardial cell hypertrophy, and mRNA levels of ANF and β-MHC in WT mice but not in NBP1-KO mice.Conclusions: These findings suggest that nobiletin inhibits cardiomyocyte hypertrophy and the development of heart failure through the functional regulation of NBP1 activity and may be a candidate for heart failure agent in human.

P08-8Heparin mobilizes human multipotent mesoangioblasts from the heart during cardiac catheterizationYoshihiro Hata1),�Masayoshi� Iwasaki1),�Keita�Horitani1),�Hiroshi�Kishimoto1),�Kensaku�Wada1),�Ichiro�Shiojima1)

1）Department of Medicine II, Kansai Medical University We previously identified circulating mesoangioblasts (cMABs), a subset of mesenchymal stem cells that co-express KDR and Nkx2.5, from the patients undergoing cardiac surgery with cardiopulmonary bypass. cMABs are capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. More recently, we demonstrated that hepatocyte growth factor (HGF) induces cMAB mobilization in animal model. We therefore tested the hypothesis that heparin induces cMAB mobilization in humans by increasing the serum levels of HGF. We also explored the niche of cMAB. Patients undergoing cardiac catheterization were treated with an increasing dose of heparin at 100U/kg (n=7), 200 U/kg (n=11) and 300 U/kg (n=11). Mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation and cultured on a fibronectin-coated dish, and the number of outgrowing colonies was counted. Heparin dose-dependently increased serum HGF levels (28.1±3.0, 34.4±1.9, and 42.8±0.9 ng/ml, respectively). Moreover, the number of cMAB colonies was also increased by heparin in a dose-dependent manner (0.27±0.26, 0.33±0.11, and 0.75±0.22 colonies/MNCs (x106), respectively). No cMAB colony outgrowth was observed in control subjects. Time course analysis of serum HGF levels and the extent of cMAB mobilization revealed that HGF concentration positively correlated with the number of cMAB colonies. cMABs mobilized by heparin were negative for hematopoietic marker CD45 and positive for mesenchymal marker CD73, the HGF-receptor c-Met, endothelial marker KDR, cardiac mesoderm marker Nkx2.5, and pluripotency markers (Oct3/4, KLF4, and cMyc). Simultaneous blood sampling from aortic sinus, coronary sinus and right atrium revealed that human heart is dominant niche of cMABs. The availability of cMABs in our modified protocol was 90.2% in 51 consecutive patients. In contrast to the availability of previously described endothelial progenitor cells, cMABs availability was not significantly affected by the presence of coronary risk factors or end-stage renal disease. In conclusion, heparin induces the mobilization of heart-derived multipotent MABs in human circulation presumably by increasing the serum HGF levels.

P09-1Identification and arrhythmogenic significance of a novel TMEM168 mutation in Brugada syndromeDimitar Zankov1),�Hisakazu�Ogita1)

1）Shiga University of Medical Science

Sudden cardiac death (SCD) is still frequent mortality cause despite the therapeutic advances in clinical cardiology. Brugada syndrome (BrS) is inherited channelopathy characterized by typical electrocardiographic (ECG) pattern, and is responsible of near 20% of SCD in the non-structural cardiac disease group. Most often BrS patients possess loss of function mutations in sodium channel subunits or associated proteins. However, in about 70% of BrS patients, causal gene mutation(s) remains unknown. We investigated a family with clinically diagnosed BrS using exome-wide sequencing, and identified heterozygous 1616 G>A substitution in TMEM168 (p.539Arg>Glu, predicted to be functionally damaging amino acid substitution). Cloned human TMEM168 (both wild-type and mutant) showed typical nuclear membrane localization in transfected COS7 cells and HL-1 cardiomyocytes, and co-localized with lamin A/C. Whole-cell patch-clamp recordings of endogenous sodium currents in HL-1 cells revealed significant decrease in sodium current density in the cells expressing mutant TMEM168, compared with wild-type TMEM168. In addition, in HL-1 cells transfected with mutant TMEM168, the amount of Nav1.5 protein, the α-subunit of cardiac sodium channel, was reduced. This reduction was caused by, at least, posttranslational regulation of Nav1.5. We then generated heterozygous TMEM168 knock-in mice that hold the same amino acid substitution as shown above in the studied BrS family, using the CRISPR/Cas9 genomic editing tool in order to evaluate cardiac electrophysiology in vivo. At the physiological state, TMEM168 knock-in mice had normal ECGs. Application of phenylephrine combined with caffeine induced various rhythm and conduction disorders, including ventricular tachycardia/fibrillation, in TMEM168 knock-in, but not in wild-type mice. Presented results associate TMEM168 mutation with arrhythmogenesis in a family with inherited BrS because of the reduction of sodium channel function.

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P09-5Gene Expression Landscape of Heart Failure Mice model caused by Cardiac Specific PRMT1 DeficiencyWeizhe Lu1）,�Kazuya�Murata2）,�Hayase�Mizukami3）,� Jun-dal�Kim4）,�Junji�Ishida4）,�Akiyoshi�Fukamizu1,3,4）

１）Ph.D. Program in Human Biology, School of Integrative Global Majors (SIGMA), University of Tsukuba, 2）Department of Biomedical Chemistry, School of Science and Technology, Kwansei Gakuin University, 3）Graduate School of Life and Environmental Sciences, University of Tsukuba, 4）Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of TsukubaHeart failure (HF) is one of sever cardiovascular diseases of which five-year survival diagnosis after a myocardial infarction has grown up to 61% (2001-2010) according to the report of American Heart Association. The pathogenesis of HF is complicated and not well understood. Recently, a growing body of evidence indicates arginine methylation (AM) which is one of the post-translational modifications is closely related to heart diseases. The enzyme expression of AM is increased in the heart of patients with coronary artery disease and the free methylated arginine is also promoted in the serum. However, the association between AM and heart is still unclear. The enzymes of AM are named protein arginine methyltransferases (PRMTs). Among all PRMTs, PRMT1 exhibited the predominant PRMTs activity. In this study, to clarify the role of AM in heart diseases, we generated the cardiomyocyte specific PRMT1 deficient (PRMT1-cKO) mice. PRMT1-cKO mice showed lethal heart failure and died within three-months of age. It is reported that the substrates of PRMT1 are various type of proteins such as transcription factors, splicing factors and ribosome proteins. We hypothesized that absence of PRMT1 affects the transcription and translation system that lead to disorder of key gene expression in cardiomyocyte. To monitor the transcription and translation, we applied the next-generation sequence. For transcription, we performed the RNA-seq that can evaluate whole mRNA expression level. Regarding to translation, we performed a cutting-edge technique named ribosome profiling (Ribo-seq) in which only the on-going translating mRNA are measured. Ribo-seq is able to provide accurate approximation for estimating the expression level of genes. By combing the results of both RNA-seq and Ribo-seq, it was revealed that the mitochondria function, glutathione metabolism purine ribonucleoside metabolic process could be impaired in the heart of PRMT1-cKO mice. We further confirmed that ATP synthesis activity was decreased in the heart of PRMT1-cKO by ATP assay.

P09-2Overlapping Brugada syndrome associated with SCN5A mutations in patients with sick sinus syndromeAsami Kashiwa1),� Takeru�Makiyama1),� Seiko�Ohno2),� Yimin�Wuriyanghai1),�Yuta�Yamamoto1),�Minoru�Horie3),�Takeshi�Kimura1)

1）Department of Cardiovascular Medicine, Kyoto University, 2）Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, 3）Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical ScienceIntroduction. The clinical spectrum of cardiac sodium channelopathies caused by mutations in SCN5A is broad, including Brugada syndrome (BrS), long QT syndrome, sick sinus syndrome (SSS), dilated cardiomyopathy, atrial fibrillation, and overlap syndromes. The objective of this study is to analyze the impact of phenotypic overlap of SSS in patients with BrS carrying SCN5A mutations on their clinical features and risk of lethal arrhythmic events.Methods. We investigated 39 patients (age 33.5±19.7 years, male 87.2%) with spontaneous or drug-induced type1 ECG pattern of BrS in whom SCN5A mutations were identified.Results. Sixteen patients (41.0%) were symptomatic with a history of syncope or ventricular fibrillation. Seven patients (23.3%) were affected by atrial fibrillation. Six of 39 patients (15.0%) initially presented SSS requiring pacemaker implantation (PMI) before the diagnosis of BrS. Between BrS patients with (n = 6) and without (n = 33) PMI due to symptomatic SSS, there were no differences in the onset age (41.0±22.0 y .o . vs . 31.0±19.4 y .o . , p = 0 .3 ) and electrocardiogram indexes (PQ interval [182.3±27.5 ms vs. 174.9±34.4 ms, p = 0.6], QRS interval [117.5±15.2 ms vs. 120.5±34.5 ms, p = 0.8], QTc interval [407.7±13.7 ms vs. 400.0±43.0 ms, p = 0.7]). Family history of SSS was more frequently observed in BrS with PMI due to SSS than without (50.0% vs. 6.1%, p = 0.02). Three of 6 patients (50%) with PMI due to SSS presented ventricular tachyarrhythmia events in 3 years, 9 years, and 10 years after PMI respectively, leading to be diagnosed as BrS and SCN5A mutation carriers (R1623X, Q779X, and S910L, respectively).Conclusions. The presence of SSS is not rare in BrS patients with SCN5A mutations. Moreover, SSS can precede the first clinical manifestation of type 1 ECG pattern of BrS in SCN5A mutation carriers, followed by life-threatening arrhythmias.

P09-3The Ref/Alt imbalance of the VUS in RNAseq affect the gene expression in cardiomyopathyYohei Miyashita1,2),�Yoshihiro�Asano1),�Hidetaka�Kioka1),�Tomohito�Otani1),�Yoshiki�Sawa4),�Seiji�Takashima3),�Yasushi�Sakata1)

1）Osaka university Dept. of Cardiovascular Medicine, 2）Osaka university Dept. of Legal Medicine, 3）Osaka university Dept. of Medical Biochemistry, 4）Osaka university Dept. of Cardiovascular SurgeryBACKGROUND: In Whole Exome Sequencing (WES), there are many Variants of Unknown Significance (VUSs), which may sometimes affect the expression difference between each allele. However, the relationship among Ref/Alt proportion, depth of its position and total gene expression level have not been fully elucidated, yet. OBJECTIVE: Focusing on the variants detected by WES, we evaluated, in regard to RNA-seq, how the Ref/Alt ratio and the total depth at each variant position affected FPKM.METHODS: Among 41 cardiomyopathy patients who underwent cardiac transplant due to severe heart failure at the Osaka University Hospital, we conducted WES from leukocytes and RNA-seq from recipient heart. We selected 257 cardiomyopathy-related genes, and detected heterozygous variants with MAF<0.5%. The relative read expression level (RRE) of a patient’s variant was defined as the quotient value obtained by dividing the read count of its position by the mean read count of the same position of controls. We assessed Ref/Alt imbalance, the variant position, RRE of the variant, and FPKM were examined following to further study whether VUSs could be stratified. RESULTS: Heterozygous variants with MAF<0.5% extracted with WES showed significantly biased towards Ref (P<0.05) in RNAseq. Plotting all variants with Ref dominancy and the RRE as the axis shows that in the high RRE group and the Ref dominance group, there were significantly disease-causing variants compared with the control group(P <0.01).The table of Ref dominancy, variants position, RRE, and FPKM for each gene was prepared, and multiple regression analysis was performed on these four elements to calculate the partial regression coefficient for FPKM. We found some VUSs significantly correlated with FPKM. DISCUSSION: When we checked a known variant on the first exon analyzed this time, despite heterozygous in WES, ref rate was 100% and RRE was decreased. Although this variant was reported as a variant that causes cardiomyopathy due to loss of function, in this analysis it was considered possible to haploinsufficiency.CONCLUSION: In addition to the variant extraction and FPKM, It was suggested that some VUSs could stratify by focusing on the read expression level and the imbalance of Ref/Alt.

P09-4The validation of association of a rare variant in TKS5 with atrial fibrillation (AF) and examination of its role in AF pathogenesisXiaoxi Yang1）

1）Bio-informational Pharmacology, Tokyo Medical and Dental University, JapanGenome-wide association study (GWAS) identified many disease-sensitive single nucleotide polymorphisms (SNPs) in various common diseases, including atrial fibrillation (AF). However, GWAS data could not be fruitful in drug discovery, which is partly due to relatively low odds ratio and localization of SNPs mostly outside of protein-coding region. To overcome this GWAS-related problems, we performed exome-wide association study (ExWAS) with collaboration with RIKEN, which identified one rare variant highly associated with atrial fibrillation (AF) (OR=3.617, P<0.0001). This variant locates in the protein-coding region of TKS5 gene and produces replacement of amino acid. We then conducted a validation trial to replicate this association in 3378 Japanese individuals with AF by genotyping. We found that the frequency of this rare variant in AF population is 10 fold higher than that in general population. When we cited genotyping data of East Asia group in 1000 genomic project, the association became statistically significant.TKS5 was originally identified as a c-Src substrate; it relates to tumor metastasis and angiogenesis by its role in formation of invodopodia as an adaptor. In AF, it is well described that macrophages migrate and infiltrate in atrial tissue, and promote local inflammation and atrial tissue degeneration, resulting in facilitation of AF. Macrophage infiltration involves invodopodia-like structure, podosome formation. Thus, we next examined the role of TKS5 in macrophage migration and infiltration. A murine macrophage cell line RAW264 was exposed to phorbol 12-myristate 13-acetate (PMA), a PKC activator, which was able to induce formation of podosomes. PMA treatment upregulated expression of Tks5. Scratch assay showed that cell migratory ability was elevated, and cell adhesion surface area was enlarged. Our data showed important role of AF-sensitive molecule, Tks5, in macrophage migration and infiltration. Experiment examining influence of AF-sensitive SNP of Tks5 on macrophage migration and infiltration is currently underway.


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P09-6Establishment of Fabry cardiomyopathy model and its isogenic control from a female patientYuki Kuramoto1),�Atsuhiko�Naito2),�Jong-Kook�Lee1,4),�Shigeru�Miyagawa5),�Yoshiki�Sawa5),�Yasushi�Sakata1),�Issei�Komuro3)

1）Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2）Department of Pharmacology, Faculty of Medicine, Toho University, 3）Department of Cardiovascular Medicine, the University of Tokyo Graduate School of Medicine, 4）Department of Advanced Cardiovascular Regenerative Medicine, Osaka University Graduate School of Medicine, 5）Department of Cardiovascular Surgery, Osaka University Graduate School of MedicineFabry disease is an X-linked disease caused by mutations in alpha-galactosidase A (GLA) gene and is characterized by defective GLA activity, globotriaosylceramide (Gb3) accumulation and organ dysfunction including Fabry cardiomyopathy. The outcomes of existing therapies are still limited and further approaches to improve the prognosis are required. In the present study, we generated induced pluripotent stem cell (iPSC) lines from female Fabry disease patient and investigated the disease-specific phenotype in iPSC-derived cardiomyocytes (iPS-CMs). We found that both GLA activity-normal and GLA activity-deficient iPSC clones could be established, suggesting that X chromosome inactivation in the somatic cell was maintained during the reprogramming process. Reactivation of the inactivated X chromosome in human female iPSC has been an issue for studying X-linked diseases; however, we found that mono-allelic expression of GLA gene was maintained for a substantial period in certain clones. Immunofluorescent staining and mass spectrometric analysis revealed that GLA activity-deficient iPS-CMs exhibited g rea t e r Gb3 a ccumu l a t i on . Ce l l u l a r hyper t r ophy and electrophysiological change were not observed, however, NPPA gene expression was increased in GLA activity-deficient iPS-CMs, indicating that “molecular hypertrophy” was readily observed in Fabry iPS-CMs. We also established a high-content analysis protocol for high-throughput screening that could quantitatively analyze Gb3 accumulation in an unbiased manner. Our screening system may help discover new drugs that would improve the prognosis of Fabry cardiomyopathy.

P09-7Involvement of Attenuation of β-Adrenoreceptors Signaling in Development of Takotsubo SyndromeTomoya Nakano1),�Kenji�Onoue1),�Kinta�Hatakeyama2),�Hiroyuki�Okura1),�Yasuhiro�Sakaguchi1),�Yoshihiko�Saito1)�1）Department of Cardiovascular Medicine, Nara Medical University, 2）Department of Pathology, Nara Medical University

Introduction: Previous reports showed that increased sympathetic nervous system (SNS) activation is related to the development of takotsubo syndrome (TTS) and dilated cardiomyopathy (DCM). However, there have been no pathological evidence reflecting difference in the characteristics of SNS activation between TTS and DCM hearts. We focused on G-protein coupled receptor kinase 2 (GRK2) and β-arrestin2, which contribute to the desensitization of β-adrenoreceptors (β-ARs). In this study, we investigated the difference of expression of GRK2 and β-arrestin2 in TTS and DCM patients, and then investigated the involvement of attenuation of β-ARs signaling in TTS development.Methods: The pathological features of 26 TTS patients (TTS group), 26 DCM patients (DCM group) and 16 normal control patients (NC group) were analyzed using left ventricular (LV) endomyocardial biopsy specimens. We investigated the expression and localization of GRK2 and β-arrestin2 in cardiomyocyte, and the degree of the phosphorylated cAMP responsive element binding protein (pCREB) to evaluate β-ARs signaling.Results: In the TTS and DCM groups, GRK2 and β-arrestin2 were observed significantly more than NC group, distributing not only in the cytoplasm but also on the cell membrane. The percentage of GRK2 and β-arrestin2 positive stained area were significantly greater in the TTS group both in the cytoplasm and on the cell membrane. However, the fraction of pCREB-positive cardiomyocyte nuclei was significantly lower in the TTS group than in the DCM group, which reflected attenuation of β-ARs signaling.Conclusions: Those pathological results indicated that transient desensitization of β-ARs followed by the attenuation of β-ARs signaling are associated with the development of TTS.

P09-8Single cell analysis of cardiomyocyte reveals spatial and temporal heterogeneity in heart failureMasahiro Satoh1,2),� Seitaro�Nomura2,3）,� Takanori� Fujita2）,�Toshiyuki�Ko3）,�Takashige�Tobita4）,�Kanna�Fujita3）,�Masamichi�Ito3）,�Hirotaka�Ieki3）,�Shintaro�Yamada3）,�Bo�Zhang3）,�Xiaohui�Tian3）,�Yoshio�Kobayashi1）,�Hiroyuki�Aburatani2）,�Issei�Komuro3）

1）Department of Cardiovascular Medicine, Chiba University Graduate School of Medicine, 2）Genome Science Laboratory, Research Center for Advanced Science and Technology, The University of Tokyo, 3）Department of Cardiovascular Medicine, The University of Tokyo, 4）Department of Cardiology, Tokyo Women's Medical UniversityIntroduction: Cardiomyocyte remodeling provoked by pathological stimuli leads to cardiac hypertrophy and subsequent heart failure, but its underlying mechanisms remain elusive.Methods and results: By integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state, and heart function, we elucidated the distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure. Single-cardiomyocyte RNA-seq analysis of the mouse heart after pressure overload showed a transcriptional heterogeneity, reconstructed a trajectory of cardiomyocyte remodeling, and revealed the dynamics of the gene programs. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Computational analysis using single-cardiomyocyte transcriptomes also unveiled a spatial transcriptional heterogeneity of Myh7, a fetal-type myosin gene. Single-molecule RNA in situ hybridization with high-throughput imaging analysis validated that Myh7 expression levels were increased and became progressively heterogeneous after pressure overload and that there was a layer-to-layer difference of the hypertrophied left ventricle, where Myh7-expressing cardiomyocytes appeared most frequently at the middle layer of the heart compared with the inner or outer layer. By integrating the morphological and transcriptomic profiles, we found that Myh7-expressing cardiomyocytes are small in size and are weak in the expression of mitochondrial translation/metabolism genes, suggesting a link between fetal gene reactivation and metabolic conversion.Conclusions: Transcriptome-based single-cell analysis uncovered the spatial and temporal heterogeneity of cardiomyocytes during heart failure. Our study suggests that cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and metabolic phenotypes.

P10-1Maternal high-fat diet promotes the expansion of abdominal aortic aneurysm in adult offspring.Makoto Saburi1),�Hiroyuki�Yamada1),�Takeshi�Sugimoto1),�Naotoshi�Wada1),�Shinichiro�Motoyama1),�Noriyuki�Wakana1),�Satoaki�Matoba1)

1）Department of Cardiovascular Medicine Kyoto Prefectural University of Medicine[BACKGROUND]Maternal high-fat diet (HFD) has been shown to modulate vascular function and remodeling in adult offspring; however, the effect on abdominal aortic aneurysm (AAA) formation remains unclear.[METHOD AND RESULT]Eight-week-old female wild-type mice (C57BL/6) were fed a HFD or normal diet (ND) one week prior to mating, and received during pregnancy and lactation. Eight-week-old male offspring of both groups underwent laparotomy and abdominal aorta from just below the left renal vein to the bifurcation was isolated. Periaortic application of 0.5 M calcium chloride (CaCl2) was administered for 15 min. Offspring of HFD-fed dams (O-HFD) showed significant increases in maximum outer diameter of AAA at 4 and 8 weeks after surgery by 22.2% and 17.6%, respectively (P<0.05), compared with offspring of ND-fed dams (O-ND). The lengths of outer circumference assessed by histological analysis were also increased in O-HFD (28.0% and 15.5%, respectively, P<0.05). Fluorescent image of abdominal aorta taken by IVIS at 7 days after surgery revealed a 2-fold increase in MMP activity (P<0.01). Consistently, the percentage of MMP-9/F4/80 double-positive cells at day 7 after surgery was significantly increased by 75.2% (P<0.01) in O-HFD versus O-ND, although the number of F4/80-positive cells was equivalent between the two groups. Intriguingly, F4/80-positive cells in O-HFD showed a 2.5-fold increase in co-staining with tartrate-resistant acid phosphate (TRAP), osteoclast marker, while TNF-α-positive staining was equivalent. To further examine the in vitro polarization to osteoclast-like macrophages, bone marrow-derived macrophages (BMDMs) were stimulated by TNF-α and M-CSF. Percentage of TRAP-positive cells was markedly increased in BMDMs form O-HFD versus O-ND (12.5% vs 6.3%, P<0.01), while LPS-induced mRNA expressions of proinflammatory cytokines were equivalent between the two groups. [CONCLUSION]Our findings show for the first time that maternal HFD promotes CaCl2-induced AAA expansion, accompanied by augmented MMPs activity. Maternal HFD-mediated skewing of macrophages toward osteoclast-like cells could be a potential therapeutic target for preventing AAA formation in adult offspring.

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P10-5MAIR-II deficiency prevents excessive inflammation and fibrosis post-myocardial infarctionSaori Yonebayashi1),�Kazuko�Tajiri1),�Nobuyuki�Murakoshi1),�Satoshi�Sakai1),�Akira�Shibuya2),�Kazutaka�Aonuma1),�Masaki�Ieda1),�Dong�Zhu�Xu1),�Fumi�Yamagami1),�Yuta�Okabe1),�Duo�Feng1),�Rujie�Qin1)

1）Department of Cardiology, University of Tsukuba, 2）Department of Immunology, University of TsukubaBackground: MAIR-II (Myeloid-Associated Immunoglobulin-Like Receptor II) is expressed on myeloid cells and is involved in Toll-like receptor-4 (TLR4)-mediated inflammatory monocyte migration from the blood to sites of infection as part of the host response in polymicrobial peritonitis. In myocardial infarction (MI), monocytes are known to be recruited to infarcted myocardium then a response of inflammatory M1 and anti-inflammatory M2 macrophages occurs. However the role of MAIR-II in MI remains elusive. Purpose: To determine the role of MAIR-II in MI pathophysiology. Methods and Results: Flow cytometric analysis revealed that MAIR-II+ myeloid cells were found to be abundant from post-MI days 3 to 5 in infarcted hearts induced by permanent ligation of the left coronary artery. To address the role of MAIR-II in myeloid cell function in vivo, effects of MAIR-II deficiency were investigated. The survival rate was significantly higher in MAIR-II knockout (KO) mice than in WT mice 30 days after MI (51% versus 28%, P=0.043). In echocardiography, MAIR-II KO mice had thicker left ventricle posterior walls and higher ejection fractions compared to wild-type (WT) mice. This indicates that MAIR-II deficiency leads to favorable post-MI remodeling. After further investigation, we found that MAIR-II KO hearts had less macrophage influx and more neutrophil infiltration after MI. Moreover, In MAIR-II KO mice there was less IL-1β gene expression, less TGF-β and type I collagen gene expressions, and more CD206+ M2 macrophages in the infarcted heart compared to WT. To elucidate MAIR-II’s role in macrophages, we analyzed bone marrow-derived macrophages (BMDM) from WT and MAIR-II KO mice polarized to M1 and M2 using LPS or IL-4 respectively. We found that M1 and M2 polarized BMDM from MAIR-II KO expressed less M1 and M2-related gene expressions compared to WT mice. Furthermore, in M1 polarized MAIR-II KO BMDM, attenuated inflammation in activated monocytes expressed by NLRP-12 was higher than that of WT. Conclusion: MAIR-II is involved in MI pathophysiology, through exacerbating inflammation and fibrosis.

P10-2“Human” epicardial adipose tissue induces “rat” atrial myocardial fibrosis through paracrine effectsShintaro Kira1),� Ichitaro�Abe1),� Yumi� Ishii1),� Takahiro�Oniki1),�Yasushi�Teshima1),�Naohiko�Takahashi1)

1）Department of Cardiology and Clinical Examination, Oita University

Background : Using excised human left atrial appendage, we demonstrated epicardial adipose tissue (EAT) are associated with atrial myocardial fibrosis as a substrate of atrial fibrillation. However, the mechanism is not clear. The purpose of this study is to clarify the mechanisms underlying the effect of EAT on the atrial myocardium. In the first stage, we observed the EAT-induced atrial myocardial fibrosis using organo-culture system. Methods: From 6 consecutive autopsy cases, human peri-left atrial EAT and abdominal subcutaneous adipose tissue (SAT) were obtained. Fat were quickly washed with PBS and cultured overnight. After pre-incubation, EAT and SAT were cultured in medium without FBS for 24hours. Finally, we collected these conditioned medium and used for experiments. To study the effects of conditioned medium, we established organo-culture system. Isolated atrium from 8-week old male Sprague-Dawley rats were placed on the porous membrane with the endothelial face toward the membrane. Then, conditioned medium or culture medium were dropped onto the epicardial face of the atrium once a day and incubated for 7days. Finally, atria were fixed with 4% paraformaldehyde, and cut into 5 μ m sections. Fibrosis were evaluated by Masson's trichrome staining. Left atria of autopsy heart also fixed and stained with Masson's trichrome staining.Results: Atria organo-culture incubated with EAT Conditioned medium showed global fibrosis. At epicardial side, fibrotic area of EAT group was significantly greater than that of SAT and control group (P<0.05). And we observed that there was fibrosis area around the epicardial fat in autopsy left atrial. Conclusions: This is the report to demonstrate the profibrotic effects of EAT and SAT on atrial myocardial fibrosis using organo-culture system. Our results clearly demonstrated that EAT rather than SAT induces atrial myocardial fibrosis through its paracrine effects.

P10-3NLRP3 Inflammasome Initiated through CaMKIIδ in Cardiomyocytes is Essential for Cardiac RemodelingTakeshi Suetomi1,2),�Shigeki�Miyamoto2),�Joan�Heller�Brown2)

1）Yamaguchi University, 2）University of California San DiegoIntroduction and Hypothesis: Inflammation is associated with cardiac remodeling in response to non-ischemic stress, but how it is initiated in the absence of cell death is not known. We tested the hypothesis that activation of CaMKIIδ in cardiomyocytes initiates inflammatory responses in response to pressure overload leading to adverse cardiac remodeling.Methods and Results: We generated cardiomyocyte specific CaMKIIδ knockout mice (CKO). CKO and control fl/fl mice (CTL) were subjected to transverse aortic constriction (TAC). CaMKII activity and NFkB activation were significantly increased in CTL but not in CKO hearts after TAC. Cardiac mRNA levels for pro-inflammatory chemokines (MCP-1, MIP1α and IL-6) and cytokines also increased and peaked at 3 days with 5 to 20 fold increases vs sham. These responses were decreased in the CKO mice. Apoptotic and necrotic cell death were absent at this time. NLRP3 mRNA levels and caspase-1 activity were increased by 3 days of TAC in isolated cardiomyocytes of CTL and to a lesser extent in CKO. CD68+ macrophage accumulation by 14 days of TAC was attenuated in the CKO and NLRP3 inhibitor MCC950 treated mice. Cardiac fibrosis, increased by 28 days TAC was significantly attenuated in the CKO and MCC950 treated mice. Ventricular dilation at 42 days was also attenuated in the CKO and MCC950 treated mice vs CTL (LVDd 4.0 ± 0.4 mm, 3.9 ± 0.5 mm vs 4.4 ± 0.5 mm), and ejection fraction was less impaired in the CKO and MCC950 treated mice vs CTL (44 ± 7%, 44 ± 5% vs 34 ± 4%). Cardiac-specific CaMKII deletion by AAV9-Cre injection in CTL prior to TAC deleted cardiac CaMKII and ameliorated inflammasome activity, macrophage infiltration, fibrosis and heart failure. On the other hand, AAV9-Cre injection at day 10 after TAC did not bring those benefits.Conclusions: Activation of CaMKIIδ in response to pressure overload triggers inflammatory gene expression and activation of the NLRP3 inflammasome in cardiomyocytes. These responses provide signals for immune cell recruitment, fibrosis and myocardial dysfunction. Early inflammatory responses induced by cardiomyocyte CaMKIIδ could be a promising target to prevent progression to heart failure.

P10-4Involvement of VGLL3 in TGF-beta-induced epithelial-to-mesenchymal transitionNoritaka Yamaguchi1,2),�Yuki�Takakura2),�Ayumi�Sugino2),�Naoto�Yamaguchi2),�Hiroyuki�Takano1)

1）Department of Molecular Cardiovascular Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, 2）Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba UniversityMyocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates various cellular processes including cell differentiation, proliferation, and epithelial-to-mesenchymal transition (EMT) through activation of the transcription factor Smad3. Because TGF-beta-induced EMT is one of the causes of myocardial fibrogenesis, understanding of the molecular mechanisms of TGF-beta-induced EMT is important for the treatment and prevention of myocardial fibrosis.To understand the molecular mechanisms of TGF-beta-induced EMT, we analyzed gene expression profiles in TGF-beta-stimulated human lung cancer A549 cells and identified genes whose expression was up-regulated by TGF-beta stimulation. We selected several genes whose roles in EMT were unclear and then established A549 cell lines stably expressing these genes. Stable expression of one of the genes, encoding the transcriptional co-factor VGLL3 (vestigial-like family member 3), was found to repress expression of the epithelial marker E-cadherin and enhance expression of the mesenchymal marker SNAIL in A549 cells. Stable expression of VGLL3 also increased cellular mobility in wound-healing assays. We next performed RNAi-mediated depletion of VGLL3 in A549 cells and analyzed its involvement in TGF-beta-induced EMT. Depletion of VGLL3 repressed change in expression of E-cadherin and SNAIL proteins and induction of cellular mobility upon TGF-beta stimulation.Finally, we analyzed the role of VGLL3 in Smad3 activation using the Smad3 reporter assay and found that VGLL3 expression stimulates activation of the Smad3 reporter. These results suggest that VGLL3 promotes EMT progression upon TGF-beta stimulation through activation of Smad3. Analyses of the molecular roles of VGLL3 in TGF-beta-induced EMT and its involvement in myocardial fibrogenesis are currently underway.


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P10-6The role of cellular senescence in cardiac fibrosis after myocardial infarctionMasato Shibamoto1),�Atsuhiko�Naitoh2),�Tomoaki�Higo1),�Shungo�Hikosoh1),�Jong-Kook�Lee1),�Yasushi�Sakata1),�Issei�Komuro3)

1）Department of Cardiovascular Medicine,Osaka University Graduate School of Medicine, 2）Department of Pharmacology, Faculty of Medicine, Toho University, 3）Department of Cardiovascular Medicine, The University of Tokyo Graduate School of MedicineIntroduction: Activation and proliferation of cardiac fibroblasts (CFs) play an important role in the formation of cardiac fibrosis after myocardial infarction (MI). Molecular mechanisms that promote these are well studied and considered as therapeutic targets for preventing excessive cardiac fibrosis. However, the molecular mechanisms by which the activation of CFs is “extinguished” and returns to inactive, non-proliferative state after MI remain unclear. Recent reports suggest that activation of DNA damage response (DDR) and cellular senescence in CFs limits fibrosis of various tissues. Methods and Results: We generated a mouse model of MI and found that the number of proliferating CFs was increased up to day 4 but gradually decreased and returned to the basal level at day 7 after myocardial infarction (MI). In contrast, the number of the CFs positive for γH2AX, an active DDR marker, and senescence-associated beta-galactosidase-positive CFs were increased up to day 7 after MI, suggesting that cellular senescence occurred in CFs at day 5-7 after MI. To test the role of DDR and cellular senescence in cardiac fibrosis, we next established an in vitro model of senescent CFs by gamma-irradiation. Co-culture of senescent CFs and non-senescent CFs revealed that senescent CFs suppress the proliferation of the surrounding non-senescent CFs not in a paracrine manner but in a contact-dependent manner. We finally generated ATM heterozygote mice to partially block DDR and prevent cellular senescence. Following MI, CFs of ATM heterozygote mice remained proliferative even after day 7 and cardiac fibrosis was aggravated compared with wild-type mice. Conclusions: Activation of DDR and cellular senescence in CFs limits cardiac fibrosis after MI through suppressing the proliferation not only of themselves but also of the surrounding non-senescent CFs. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.

P10-7Identification of Cardiovascular Dysfunction in Spondyloarthritis Model RatsHiroki Hayashi1),� Jiao�Sun1),�Tetsuya�Tomita1),�Hironori�Nakagami1)

1）Osaka University

The spondyloarthritis (SpA) has been clinically known to be related to cardiovascular diseases, including valve disease, aortitis, conduction disturbances. Although Taurog et al. developed HLA-B27-Transgenic rats mating with human β2-microglobulin as a SpA model, cardiovascular system has not been well investigated in this model. The final goal of our research is to identify and evaluate cardiovascular dysfunction in HLA-B27 tg rats to reveal how SpA links to cardiovascular abnormalities. Based on the previous paper by Taurog et al., we established transgenic rats by crossbreeding between 21-3 rat transgenic line (with 20 transgene copies of HLA-B27 and 15 transgene copies of human β2-microglobulin: hemizygous) and 283-2 rat transgenic line (with 35 transgene copies of hβ2m: homozygous). We found that 34 rats out of 54 rats has transgenes successfully (with 20 transgene copies of HLA-B27 and 50 transgene copies of human β2-microglobulin), analysed by genotyping technique. The male rat with HLA-B27/hβ2m transgenes (21-3 x 283-2 ) exhibited SpA l ike phenotype (Epididymoorchitis, Arthritis, or Spondylitis) from 100 days after birth. We evaluated the phenotypic severity based on paw thickness, tail swelling and size of epididymis every 4 weeks from 100 days to 250 days. Scoring evaluation, X-ray and immunohistological analysis suggested that male HLA-B27/hβ2m transgenic rats (21-3 x 283-2) had severe arthritic phenotype. At this stage, we extracted heart and aorta to evaluate the manifestation of cardiovascular abnormalities by histological techniques (HE staining, elastin van Gieson staining). As the results, we identified abnormalities in cardiac tissue and aorta in HLA-B27 tg rat. In conclusion, we found that SpA-related cardiovascular dysfunction in HLA-B27 tg rat for further investigations to clarify the relationship between SpA and cardiovascular dysfunctions.

P10-8Beneficial effects of exercise on cardiac fibrosis in obese mice: the role of leptin.Maria Pini1),�Gabor�Czibik1),�Raquel�Mercedes1),�Julien�Ternacle1),�Corneliu�Henegar1),�Genevieve�Derumeaux1),�Daigo�Sawaki1)

1）IMRB - Inserm U955BACKGROUND: Physical activity can favor health in aging and obesity. We previously demonstrated that endocardial f ibrosis and cardiovascular dysfunction decrease in response to exercise in aged mice. Moreover exercise may prevent adipose tissue premature senescence induced by long-term high fat diet (HFD), improving the cardio-metabolic phenotype. OBJECTIVES: We aim to understand whether short-term HFD is sufficient to induce premature senescence of white adipose tissue (WAT), influencing cardiac and metabolic functions. To determine whether exercise-mediated WAT improvements are important for myocardial alterations, we evaluated the response of two different adipose depots, subcutaneous (inguinal, iWAT) and visceral (epididymal, eWAT). METHODS & RESULTS: Five-month-old male C57BL/6J were exposed to HFD for 10 weeks and evaluated in either sedentary or exercise (swimming) conditions. Excess of calories and sedentarism affected cardiac structure and function, in association with local inflammation and apoptosis. Exercise reduced myocardial collagen accumulation and fibrotic gene programs, rescuing LV global contractility (echocardiography and in vivo hemodynamic) in HFD mice. In parallel, exercise normalized the upregulation of key inflammatory markers, TNFα, IL6 and the chemokine CCL2, known to be important for the activation of fibroblasts, and abrogated cellular senescence (SA ß-galactosidase and cyclin-dependent kinase inhibitor 2A, p16) in iWAT and eWAT. In the context of obesity, one of the factors promoting cardiac remodeling is the adipokine leptin. Indeed we evidenced an association between cardiac collagen content and plasma leptin which underlines the potential critical link between HFD-induced heart and WAT dysfunctions. Exercise prevented the increased circulating leptin in HFD mice, despite no significant fat mass reduction and in association with reversed hypertrophy No major inflammatory and fibrotic responses were observed in both depots. Finally, rescue of WAT alterations by exercise was associated with restoration of glucose homeostasis in HFD mice, as demonstrated by glucose and insulin tolerance tests. CONCLUSIONS: Improvement of cardiac function and glucose metabolism by exercise reinforce the view that WAT endocrine role is critical for cardiac health in the context of obesity.

P11-1Prevalence of risk factors for CVD among children and adolescents of Asian Indian originPartha Sarathi Datta1）

１）Department of Anthropology, Visva-Bharati, Santiniketan, India.Objectives: The purpose of this cross-sectional study was to find out the prevalence of cardiovascular disease (CVD) risk factors in school going children and adolescents of Asian Indian origin. Materials and Methods: This study was carried out among 1101 (532 boys and 569 girls) children and adolescents, aged 10 to 17 years from rural, suburban and urban schools in West Bengal, India. Anthropometric measurements, such as stature, body weight, circumferences at mid arm (MUAC), minimum waist (MWC) and maximum hip (MHC), skinfolds at biceps (BSF), triceps (TSF), sub scapular (SSSF) and supra iliac (SISF) regions etc., were measured using standard methodology. Systolic (SBP) and Diastolic (DBP) blood pressure were recorded according to a proper methodology. Lipid profiles, such as total cholesterol (TC), triglyceride (TG), high (HDL), low (LDL), very low-density lipoprotein (VLDL), and blood glucose were also measured from each participant. A schedule was used to collect data on the socio-demographic profile, birth records, behavioural activity, family history of blood pressure, diabetes, CVD etc. and weekly physical activities. The weekly consumption of food was collected using a food frequency schedule.Results: The urban participants have higher mean values of Body Mass Index (BMI) and Waist Hip Ratio (WHR). But significantly, mean values of systolic blood pressure & diastolic blood pressure are almost similar in participants from different habitat variation. No significant sex difference is observed for blood pressures. Mean values of blood glucose and lipid profile are slightly higher in the urban participants. Sedentary lifestyle and faulty food habits was found to be significant association with lipid profile and blood glucose level in the participants.Conclusion: The study showed that the prevalence of CVD risk factors was high in both urban, sub urban and rural participants. Prevention should begin during early ages in life, when a modification in lifestyle can reduce the incidence of CVD. Therefore, there are need an effective preventive strategy and health awareness programs, targeting the children & adolescent to encourage and improve their unhealthy life style, so that they do not become the epidemics of the 21st century.Keywords: CVD risk factors, children and adolescents, Asian Indian.

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P11-5MURC/Cavin-4 deficiency improves cardiac ischemia-reperfusion injuryYumika Tsuji1),� Takehiro�Ogata2),�Masahiro�Nishi1),�Naohiko�Nakanishi1),�Yusuke�Higuchi1),�Akira�Sakamoto1),�Satoaki�Matoba1)

1）Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, 2）Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine

Background: Ischemia-reperfusion (I/R) injury is considered as a critical issue in strategy for ischemic heart disease. Muscle-restricted coiled-coil protein (MURC)/Cavin-4 belongs to the cavin family, which is a component of caveola. We previously reported that MURC/Cavin-4 is involved in myofibrillar organization and cardiomyocyte hypertrophy. MURC/Cavin-4 mutations are observed in patients with dilated cardiomyopathy. However, the role of MURC/Cavin-4 in cardiac I/R injury remains unknown.Methods and results: I/R model of mice heart was created by 1h ligation of left anterior descending artery, followed by 24h reperfusion. TTC staining showed that MURC/Cavin-4-/- mice exhibited significantly smaller infarct size. Echocardiography showed that fractional shortening (FS) was significantly preserved in MURC/Cavin-4-/- mice. Reactive oxygen species (ROS) assessed by DHE staining in mice heart was lower in MURC/Cavin-4-/- mice. Western blotting of mice heart showed that STAT3 was significantly activated in MURC/Cavin-4-/- mice. In addition, mRNA expression of bcl-2 was significantly increased in the hearts of MURC/Cavin-4-/- mice. Moreover , TUNEL sta in ing per formed in ra t neonata l cardiomyocytes subjected to hydrogen peroxide showed that the number of apoptosis cel ls was signif icantly decreased in cardiomyocytes transduced by MURC/Cavin-4 siRNA.Conclusion: MURC/Cavin-4 deletion reduced cardiac I/R injury by reduction of ROS and anti-apoptotic signaling with STAT3 activation. MURC/Cavin-4 could be a therapeutic target for cardiac I/R injury.

P11-2Akt1-mediated muscle growth promotes blood flow recovery by enhancing HO-1 in neighboring cellsYasuhiro Izumiya1),�Yoshiro�Onoue2),�Shinsuke�Hanatani2),�Toshifumi�Ishida2),�Yuichiro�Arima2),�Kenichi�Tsujita2)

1）Osaka City University, 2）Kumamoto University

Background: Resistance exercise has beneficial effects on patients with peripheral arterial diseases. We assessed the hypothesis that muscle growth promotes angiogenesis by interacting with neighboring cells in ischemic lesions.Methods and Results: We used skeletal muscle-specific inducible Akt1 transgenic (Akt1-TG) mice that induce growth of functional skeletal muscles as a model of resistance training. Proteomics analysis identified significant upregulation of Heme oxigenase-1 (HO-1) in muscle tissue in Akt1-TG mice compared with control mice. Blood flow recovery after hindlimb ischemia was significantly increased in Akt1-TG mice compared with control mice. Enhanced blood flow and capillary density in Akt1-TG mice was completely abolished by the HO-1 inhibitor, Tin-mesoporphyrin. Immunohistochemistry showed that HO-1 expression was not increased in muscle cells, but in macrophages and endothelial cells. Consistent with these findings, blood flow recovery after hindlimb ischemia was similar between control mice and skeletal muscle-specific HO-1-knockout mice. Adenoviral-mediated overexpression of Akt1 did not increase HO-1 protein expression in C2C12 myotubes, however, the conditioned medium from Akt1-overexpressing C2C12 myotubes increased HO-1 expression in endothelial cells. Cytokine array demonstrated that a panel of cytokine secretion was upregulated in Akt1-overexpressing C2C12 cells, suggesting paracrine interaction between muscle cells and endothelial cells and macrophages.Conclusions: Akt1-mediated muscle growth improves blood flow recovery after hindlimb ischemia by enhancing HO-1 expression in neighboring cells.

P11-3Iron Metabolism in a Mouse Model of Hind Limb IschemiaKeisuke Okuno1),�Yoshiro�Naito1),�Seiki�Yasumura1),�Koichi�Nishimura1),�Masanori�Asakura1),�Masaharu� Ishihara2),�Tohru�Masuyama1)

1）Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, 2）Division of Coronary Heart Disease, Department of Internal Medicine, Hyogo College of MedicineBackground: Iron is an essential trace mineral in the living body, while excess iron causes tissue damage. Phlebotomy decreases serum iron concentration and improves clinical outcomes (cardiovascular death, etc.) in patients with peripheral artery disease (PAD). It has not been investigated whether iron contributes to the pathogenesis of PAD through angiogenesis.Objective: The aim of this study is to examine whether iron metabolism effects on angiogenesis in a mouse model of hind limb ischemia (HLI).Methods: To induce HLI, the left femoral artery in 8 - 9 week-old male mice was ligated and stripped, and the right hind limb was used as a control. Blood flow was measured by a laser Doppler blood flowmetry at before and after surgery day0, 3, 14, and 28. Subsequently, blood and adductor muscles were harvested for Western blot and immunohistochemistry to evaluate the protein expression related to angiogenesis.Results: The ratio of ishemic/non-ischemic hind limb blood flow was significantly decreased to 12% at baseline, and recovered to 62% at day 28. Interestingly, expression of ferritin, a maker for iron storage, in ischemic adductor muscle was decreased compared to non-ischemic adductor muscle, indicating that tissue iron was needed during angiogenesis. Since iron uptake into cell is mediated by transferrin receptor 1 (TfR1), a receptor of iron uptake into cell, we next evaluated angiogenesis in TfR1 hetero deleted mice model of HLI. TfR1 hetero deleted mice also showed similar reduction of blood flow at baseline, whereas blood flow recovery at day 28 was attenuated in TfR1 hetero deleted mice compared to wild type mice. Ferritin expression in ischemic adductor muscle was decreased in TfR1 hetero deleted mice compared to wild type mice.Conclusions: Iron metabolism is associated with the process of angiogenesis in a mouse model of HLI.

P11-4AIM Depletion Suppressed Cardiac Rupture without Affecting Hemodynamics after MI in MiceShohei Ishikawa1),�Takahisa�Noma1),�Makoto� Ishizawa1),�Kaori� Ishikawa1),� Teppei� Tsuji1),� Kazushi�Murakami1),�Tetsuo�Minamino1)

1）Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa UniversityBackground Apoptosis inhibitor of macrophage (AIM) is secreted specifically by macrophages and contributes to macrophage accumulation in inflamed tissues. Cardiac rupture is an important cause of death in the acute phase after myocardial infarction (MI). First MI, anterior infarct, female sex, hypertension, and elderly are risk factors for cardiac rupture. Pathologically, macrophages play a pivotal role in cardiac remodeling and cardiac rupture after MI. In this study, we evaluated the role of AIM in macrophage accumulation in the infarcted myocardium and cardiac rupture after MI. Methods Wild-type (WT) and AIM-knockout (KO) mice underwent permanent left coronary artery ligation and were followed-up for 7 days. Blood pressures (BP) and pulse rates (PR) were measured by the tail-cuff method. Left ventricular end-diastolic and end-systolic diameter (LVDd and LVDs, respectively) and fractional shortening (FS) were measured by transthoracic ultrasound cardiography. Hemodynamic analysis was performed for measurement of left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP). Macrophage accumulation were evaluated by immunohistology. Matrix metalloproteinase (MMP) activities were measured by zymography. Results AIM-KO mice had significantly better survival and lower cardiac rupture rate than WT mice (81.1% vs. 48.2%, P<0.05 and 10.8% vs. 31.5%, P<0.05, respectively). Before coronary artery ligation, there were no significant differences in systolic BP or PR. Following MI, LVDd and LVDs were increased, and FS was decreased in the two groups. However, there were no significant differences in these parameters between WT and AIM-KO mice. LVSP and LVEDP measured by hemodynamic analysis at 3 days after MI were also comparable between WT and AIM-KO mice. Macrophage number and MMP-2 and 9 activities in the infarcted myocardium were significantly lower in AIM-KO mice than in WT mice. Conclusions AIM depletion decreases macrophages, which are a potent source of MMP-2 and 9, in the infarcted myocardium and improves survival rates by suppressing cardiac rupture without affecting left ventricular function and hemodynamics in the acute phase after MI.


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P11-6Lack of Sirt7 attenuates myocardial ischemia-reperfusion injury by modulating NRF2 activitySatoshi Araki1),� Satoshi� Araki1),� Satoru� Yamamura1),�Kenicih�Tsujita1)

1）Department of Cardiovascular Medicine, Faculty of Life Sciences, Kumamoto University

Background: Sirtuins are NAD-dependent histon and protein deacetylase. Sirt7 is one of the seven members of the mammalian sirtuin family. Recently, we reported that Sirt7 modulates wound healing processes after myocardial infarction by regulating TGF beta receptor I expression. However, the role of Sirt7 in myocardial ischemia-reperfusion (IR) injury is not investigated. Methods and Results: Sirt7 expression was decreased in response to ischemia-reperfusion injury. Next, we created ischemia-reperfusion model in homozygous Sirt7 deficient (Sirt7-/-) mice and wild type (WT) mice. There was no difference in the area at risk (AAR). However, the infarct area/AAR were significantly decreased in Sirt7-/- mice compared with WT mice. These data suggest that lack of Sirt7 protects IR injury. In vitro experiments, Sirt7 siRNA-treatment in rat neonatal cardiomyocyte reduced hypoxia-reoxygenation induced Cleaved-Caspase3 expression. Cell apoptosis assessed by LDH assay were also decreased by sirt7 knockdown. It is reported that Nrf2 activation protects ischemia/reperfusion injury by regulating antioxidative factors.Sirt7 knockdown increased antioxidative factors, such as HO-1 and NQO-1. Luciferase assay assay revealed that NRF2 transcriptional activity was increased by Sirt7 siRNA treatment. Conclusions: Lack of Sirt7 attenuates ischemia-reperfusion injury by modulating NRF2 activity.

P11-75-Aminolevulinic Acid Combined with Ferrous Iron Ameliorates Cardiac Ischemia/Reperfusion Injury.Yu Oimatsu1),�Koichi�Kaikita1),�Masanobu�Ishii1),�Nobuhiro�Nakanishi1),�Tatsuro�Mitsuse1),�Kenichi�Tsujita1)�1）Kumamoto UniversityPost ischemia/reperfusion injury (I/R) reduces beneficial effect of restoration of coronary flow in the setting of acute myocardial infarction. 5-aminolevulinic acid (5-ALA) is known to be a naturally occurring metabolic precursor of heme, and 5-ALA combined with ferrous iron can induce heme oxygenase-1 (HO-1) in various cells. It was reported that 5-ALA combined with ferrous iron improved post renal I/R. In this study, we investigated the protective effect of 5-ALA after cardiac I/R using mice model. Male C57/BL 6J mice (8-12 weeks of age and weighing 21-26 g) were pretreated with 100 mg/kg of 5-ALA/HCL and 157 mg/kg of sodium ferrous citrate or vehicle 48, 24, 1 h before I/R, and underwent 50 minutes of left coronary artery occlusion followed by reperfusion. After 24 h of reperfusion, infarct area (IA) and area at risk (AAR) were determined by 5% Evans blue (EB) and 1% triphenyltetrazolium chloride (TTC) double staining. EB/TTC staining showed that pretreatment of 5-ALA group significantly reduced IA/AAR compared to vehicle group (34.0% vs. 51.7%, p= 0.001, n=6). Immunostaining of ischemic area after 24 h of reperfusion showed that infiltration of neutrophils and macrophages were significantly reduced in 5-ALA group than that of vehicle group. Real time PCR assay 8h after reperfusion showed that mRNA expression of TNF-α (p=0.028), IL-1β(p=0.026), TIMP-1 (p=0.026), iNOS (p=0.003), and BNP (p=0.043) were significantly lower in 5-ALA group than those in vehicle group. Moreover, the expression of mRNA of HO-1 was significantly higher than that of vehicle group (p=0.002). Western blotting assay after 8h of reperfuion showed that the protein level of HO-1 in ischemic region of 5-ALA group was significantly higher than that of vehicle group (p=0.002). To assess the HO-1 dependent protective effect of 5-ALA after cardiac I/R, we performed inhibition experiment using zinc protoporphyrin-9 (ZnPPIX), which is an inhibitor of HO-1. EB/TTC double staining after 24h reperfusion showed that the protective effect of 5-ALA was inhibited in 5-ALA/ZnPPIX group. These findings suggested that 5-ALA combined with ferrous iron might play a protective role after I/R by attenuating inflammatory reaction via increasing HO-1 expression.

P11-8Human iPSC-derived cardiac tissue transplantation to a rat unloaded ischemic heart modelDaisuke Heima1,2),�Hidetoshi�Masumoto1,3,4),�Masafumi�Takeda2),�Tadashi�Ikeda1),�Yasuhiko�Tabata5),�Kenji�Minatoya1),�Jun�Yamashita2)1）Department of Cardiovascular Surgery, Graduate school of Medicine, Kyoto University, Kyoto, Japan, 2）Department of Cell Growth and Differentiation, Center for IPS Cell Research and Application (CiRA), Kyoto university, Kyoto, Japan, 3）RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan, 4）RIKEN Center for Developmental Biology (CDB), Kobe, Japan, 5）Laboratory of Biomaterials, Department of Regeneration Science and Engineering Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, JapanBACKGROUNDS: Human induced pluripotent stem cell (hiPSC)-derived cardiac tissue sheets (CTSs) hold potentials in cardiac functional recovery. Here we investigated the effects of hiPSC-derived cardiac tissues (HiCTs) transplantation to an ischemic heart under unloading condition induced by heterotopic heart transplantation mimicking left ventricular assist device implantation. METHODS: We generated 5-layered HiCTs by inserting gelatin hydrogel microspheres between CTSs (HiCT-5). Myocardial infarction was induced in male athymic nude rats by a permanent ligation of left anterior descending artery. One week later, the infarcted heart and lung were harvested and ectopically transplanted into the abdominal aorta of a recipient nude rat. Infarcted hearts were evaluated under the 3 different conditions: heterotopic heart transplantation group (HTx), heterotopic heart transplantation with HiCT-5 transplantation group (HTx+CTS) and sham operation group (with no heterotopic transplantation) as a control (MI). Four weeks later, we histologically evaluated the grafted hearts. RESULTS: Transplanted HiCT-5 formed regenerated myocardium in 5 out of 6 rats of HTx+CTS group (83.3%). cTnT/Ki67 double positive cells were observed in the regenerated myocardial region indicating the proliferative potential of engrafted hiPSC-derived cardiomyocytes 4 weeks after transplantation. Infarcted area in HTx+CTS was significantly lower compared to those in HTx group and MI group （MI vs HTx vs HTx+CTS ：33.6±6.2 vs 34.5±8.4 vs 22.4±5.5 %; P=0.01). HTx+CTS group showed higher number of capillaries in border zone than those of HTx group and MI group (MI vs HTx vs HTx+CTS： 12.7±4.7 vs 16.0±1.6 vs 70.5±58/mm2; P=0.0025). HiCT-5 transplantation suppressed the atrophy of cardiomyocytes caused by the unloading (cross section area of cardiomyocyte: MI vs HTx vs HTx+CTS: 819.0±158.9 vs 335.6±72.2 vs 622.3±139.0 um2, P=0.001). CONCLUSION: HiCT transplantation is a potentially effective method for the treatment of ischemic hearts under the left ventricular assist device through the attenuation of ventricular remodeling, promotion of angiogenesis and cardiomyocyte proliferation, and the attenuation of cardiomyocyte atrophy, which may serve as a therapeutic strategy in the “bridge to recovery” of ventricular assist devices in the future.

P12-1The Body-wide Transcriptome Landscape of Disease ModelsKyoji Urayama1,2),�Satoshi�Kozawa1,2),�Ryosuke�Ueda1,2),�Fumihiko� Sagawa1,2), � Satsuki� Endo1,2), � Thomas� N.�Sato1,2,3,4,5)

1）The Thomas N. Sato BioMEC-X Laboratories, Advanced Telecommunications Research Institute International (ATR), Kyoto, Japan, 2）ERATO Sato Live Bio-Forecasting Project, Japan Science and Technology Agency (JST), Kyoto, Japan, 3）Karydo TherapeutiX, Inc., Tokyo, Japan, 4）Department of Biomedical Engineering, Cornell University, Ithaca, USA, 5）Centenary Institute, Newtown, Australia

Virtually all diseases affect multiple organs. However, our knowledge of the body-wide effects remains limited. Here, we report the body-wide transcriptome landscape across 13–23 organs of mouse models of myocardial infarction, diabetes, kidney diseases, cancer, and pre-mature aging. Using such datasets, we find (1) differential gene expression in diverse organs across all models; (2) skin as a disease-sensor organ represented by disease-specific activities of putative gene-expression network; (3) a bone-skin cross talk mediated by a bone-derived hormone, FGF23, in response to dysregulated phosphate homeostasis, a known risk-factor for kidney diseases; (4) candidates for the signature activities of many more putative inter-organ cross talk for diseases; and (5) a cross-species map illustrating organ-to-organ and model-to-disease relationships between human and mouse. These findings demonstrate the usefulness and the potential of such body-wide datasets encompassing mouse models of diverse disease types as a resource in biological and medical sciences. Furthermore, the findings described herein could be exploited for designing disease diagnosis and treatment.

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P12-5Gut microbiome composition and plasma microbial metabolites in patients with heart failureTomohiro Hayashi1),� Tomoya� Yamashita1),� Naofumi�Yoshida1),�Tokiko�Tabata1),�Yasuhiro� Irino1),�Ryuji�Toh1),�Ken-ichi�Hirata1)

1）Kobe University Graduate School of MedicineBackground-Gut microbiome interacts with the host physiology including immune response and systemic metabolism, and we have identified the gut bacterial flora type associated with coronary artery disease. The role of gut microbiome in the pathophysiology of heart failure (HF) has also been attracting attention. Methods and Results-We prospectively investigated gut flora by analyzing bacterial DNA extracting from patients’ stool with high-throughput sequencing of the bacterial 16S ribosomal RNA genes, and plasma microbiome-related metabolites by capillary electrophoresis time-of-flight mass spectrometry analysis at different 2 time points of hospitalized 22 HF patients both in decompensated and compensated phases. We have just proved that compensated HF patients had a significantly higher proportion of the phylum Actinobacteria compared with age- and sex- matched hospitalized 11 controls with conventional risk factors but no history of HF (p=0.027). The genus Bifiodobacterium was abundant, while Megamonas was depleted in HF patients (p=0.018 and p=0.002, respectively). Meanwhile, plasma concentration of trimethylamine N-oxide (TMAO), one of gut microbiome-associated metabolites, was significantly increased in HF patients with both reduced and preserved ejection fraction. A correlation analysis revealed positive correlations between the abundance of the genus Escherichia/Shigella and levels of TMAO and indoxyl sulfate (IS, a microbe-dependent uremic toxin) in compensated HF (TMAO, r=0.62, p=0.002; IS, r=0.63, p=0.002). Escherichia/Shigella was more abundant in decompensated than in compensated HF (p=0.030). Conclusions-Gut microbiome composition and microbiome-related metabolites are altered in HF patients and specific bacterial genera are correlated with circulating levels of harmful metabolites. Gut microbiome and their relevant metabolites could be novel biomarkers and also targets for the treatment of HF.

P12-2Purification of the growing skeletal muscle derived exosomal micro RNA.Toshifumi Ishida1),�Yuichirou�Arima1),�Yasuhiro� Izumiya2),�Masahiro� Yamamoto1),� Satoru� Yamamura1),� Satoshi�Araki1),�Kenichi�Tsujita1)

1）kumamoto university, 2）Osaka City University

Background: Supervised exercise has beneficial effects on patients with cardiovascular disease. However, underling mechanism related to the inter-organ crosstalk was not fully understood. Recently, tissue derived exosomes are focused and convey many types of signaling molecules. Recently, exosomal microRNAs (miRNAs) are paid the attention and we tried to purify the miRNAs included in growing skeletal muscles.Methods: We used skeletal muscle-specific inducible Akt1 transgenic (Akt1-TG) mice. Using the Tet-On system of gene activation, growth of skeletal muscles was induced without exercise training. Exsomes were purified combined usage of ultracentrifugation. miRNAs were extracted from purified exosomes by spin column-based method. Western blotting and real-time PCR were used for exosome and miRNA detection, respectively.Results: Akt1 gene activation was successfully performed and Akt1-TG mice showed remarkable ske leta l musc le growth (Weight o f gastrocnemius muscle: Akt1-TG (0.209±0.030)g vs Control (0.163±0.022)g, p<0.01). We obtained exsome from serum. Western blotting analysis revealed strong expression of CD9 and CD63 proteins, markers of exosome. Total amount of exosome did not show the significant difference between Akt1-TG and Control mice . Real-time PCR confirmed extraction of miRNA by RNU-6 detection.Conclusions: We successfully extracted miRNA-containing exosomes secreted from growing skeletal muscle. Total amound did not show the significant differences. In future direction, quantitative and global analysis will be required.

P12-3Gut Microbiota Depletion Alters Cardiac Hypertrophy in MiceTakehiro Kamo1),�Takako�Yao1)

1）The Institute for Adult Diseases, Asahi Life Foundation

Gut microbiota play an essential role in the maintenance of host homeostasis. There is growing evidence that an altered composition of gut microbiota, known as dysbiosis, is associated with a wide spectrum of host disorders ranging from gastrointestinal diseases to inflammatory, metabolic, hepatic, neurologic, and cardiovascular diseases. Experimental studies with fecal microbiota transplantation from human donors to germ-free animals have suggested that dysbiotic gut microbiota can reproduce host disease phenotypes in recipients. We recently showed that heart failure is associated with gut microbiota dysbiosis through 16S ribosomal RNA gene sequencing of fecal samples from patients with heart failure. This finding is suggestive of a potential significance of gut microbiota in the pathophysiology of heart failure. However, the influence of gut microbiota on the pathophysiological process of heart failure is still unknown. To test the hypothesis that gut microbiota regulate host cardiac hypertrophy, we treated mice with orally administered broad-spectrum antibiotic cocktail to deplete gut microbiota before the injection of isoproterenol. We confirmed the depletion of fecal bacterial biomass caused by broad-spectrum antibiotics, as assessed by quantitative PCR targeting 16S ribosomal RNA gene. Oral administration of antibiotics prevented death following isoproterenol injection. Antibiotic-treated mice showed a significant decrease in cardiac hypertrophy in response to isoproterenol as compared with untreated mice. In addition, oral treatment with antibiotics led to changes in sarcomere gene expression in the heart, and attenuated cardiomyocyte hypertrophy induced by isoproterenol. These results suggest that gut microbiota may contribute to isoproterenol-induced cardiac hypertrophy through the regulation of gene expression in cardiomyocytes. The novel approach targeting gut microbiota may offer a new therapeutic paradigm in the treatment of heart failure.

P12-4The alterations of gut epithelial barrier function in a mouse model of heart failureAkiko Saga-Kamo1),�Hiroshi�Akazawa1),�Masahiko�Umei1),�Hiroki� Yagi1),�Qing� Liu1),� Hiroshi�Matsunaga1),� Hiroshi�Kadowaki1),�Ryo�Matsuoka1),�Issei�Komuro1)��1）Department of Cardiovascular Medicine, The University of Tokyo Hospital

Recently, the potential role of the gut in heart failure has attracted increasing attention. We recently demonstrated the compositional alterations in gut microbiota of patients with heart failure as compared with healthy control subjects. In heart failure, decreased cardiac output and increased congestion may lead to functional and structural changes of gut, including gut epithelial barrier dysfunction. However, the mechanisms of gut epithelial dysfunction in the pathogenesis of heart failure have not been elucidated and little is known about the impact of them. We found many alterations in gut pathology characterized by villous atrophy and decreased number of goblet cells in mice with isoproterenol-induced heart failure. Also, a significant decrease in the intestinal expression levels of antimicrobial lectins, and a significant increase in the bacteria adhering to gut epithelia were found. In addition, the circulating levels of lipopolysaccharide were increased in mice with heart failure, indicating increased permeability of gut epithelial barrier and translocation of lipopolysaccharide. These results suggest that decreased expression of antimicrobial lectins in gut epithelia may compromise the gut epithelia barrier function and induce translocation of microbial product into systemic circulation, leading to the activation of inflammatory responses and exaggeration of heart failure. Our studies have profound significance in a potential intervention protecting gut epithelial barrier function against heart failure.


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P12-6Serum FABP4 is removed by the kidney via glomerular filtration and megalin-mediated reabsorptionTatsuya Iso1),� Suman� Shrestha1),� Hiroaki� Sunaga1),�Masahiko�Kurabayashi1)

1）Gunma University Graduate School of Medicine

Background Circulating fatty acid binding protein 4 (FABP4), secreted from adipocytes, is a potential biomarker for metabolic and cardiovascular diseases. Serum levels of FABP4 are positively associated with adiposity and adrenergic stimulation, but negatively with renal function. However, the mechanism underlying clearance of circulating FABP4 by the kidney remains unsolved. Methods and Results Tracing study revealed remarkable accumulation of 125I-labeled FABP4 in the kidney, but not in liver and heart, 10 min after intravenous injection. FABP4 conjugated with Alexa Fluor 647 was exclusively detected in the apical membrane of proximal tubule epithelial cells (PTECs) 10 min after intravenous injection. Bilateral nephrectomy resulted in marked elevation of circulating FABP4 in wild type mice, confirming an essential role of the kidney for clearance of circulating FABP4. Megalin, a transmembrane endocytic receptor expressed in the apical membrane of PTECs, is well-known to play a major role in reabsorption of proteins filtered through glomeruli. Quartz-Crystal Microbalance study revealed that FABP4 binds to megalin. In kidney-specific megalin knockout mice, a large amount of FABP4 was excreted to urine while serum levels of FABP4 were significantly reduced, suggesting megalin-mediated reabsorption, mostly for catabolism but partly for retrieval to the circulation. Conclusions Majority of circulating FABP4, produced and secreted from adipocytes, is eliminated by the kidney via the glomerular filtration followed by megalin-mediated reabsorption. Our data suggest that serum levels of FABP4 are determined by balance between secretion rate of FABP4 from adipocytes and clearance rate of the kidney.

P12-7Transferrin receptor 1 is associated with the development of diabetic kidney diseaseSeiki Yasumura1),�Yoshiro�Naito1),�Keisuke�Okuno1),�Koichi�Nishimura1),�Masanori�Asakura1),�Masaharu� Ishihara2),�Tohru�Masuyama1)

1）Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, 2）Division of Coronary Heart Disease, Department of Internal Medicine, Hyogo College of Medicine

<Introduction> Diabetic kidney disease (DKD) is one of the most important complications of diabetes mellitus and the leading cause of end-stage renal disease. Substantial renal iron loads have been reported in animal models of DKD. Transferrin receptor 1 (TfR1) plays a key role in cellular iron transport. However, there are no reports on the role of TfR1 in the pathophysiology of DKD. Here, we assessed the hypothesis that TfR1 is associated with the development of DKD.<Methods> To investigate an association of TfR1 in the pathophysiology of DKD, wild-type mice and TfR1 hetero knockout mice were injected with streptozotocin (STZ:50mg/kg body weight, i.p.) for five days to induce DKD. At 12 weeks after STZ injection, mice were sacrificed.<Results>Blood glucose levels were increased in wild-type and TfR1 hetero knockout mice at 12 weeks after STZ injection, and the extent was not significantly different between wild-type and TfR1 hetero knockout mice. Urinary albumin to creatinine ratio was not different between wild-type and TfR1 hetero knockout mice at 12 weeks after STZ injection. However, TfR1 hetero knockout mice showed attenuated mesangial matrix expansion and renal fibrosis, along with decreased renal expression of ferritin as compared with wild-type mice at 12 weeks after STZ injection.<Conclusions> These results indicate that TfR1 is associated with the development of DKD. Understanding the role of TfR1 in the pathophysiology of DKD may lead to a novel therapeutic approach for DKD.

P12-8Renal Tissue Hypoxia in the Subacute Phase of Renal Ischemia Reperfusion InjuryPei Chen Connie Ow1,2),�JenniferP�Ngo2),�RogerG�Evans2),�MdMahbub�Ullah2),�Giannie�Barsha2),�Lucinda�Hilliard2)

1）National Cerebral and Cardiovascular Research Institute, 2）Monash University, 3）Maastricht University, 4）University of Bristol, 5）University of ExeterIschemia-reperfusion injury (IRI) sustained from medical interventions often arises from the need to restrict or completely prevent blood flow into the kidney. The kidney is often observed to be hypoxic during ischemia and in the immediate hours following reperfusion. However, little is known about renal tissue PO2 during the extension and recovery phase of IRI. We hypothesized that tissue hypoxia is present in the subacute phase of renal IRI and could potentially play a role in the development of ischemia-induced chronic kidney disease (CKD). To induce ischemia, blood flow into both kidneys was prevented for 1 h using microvascular clamps. We implanted an oxygen telemeter into the medulla of the left kidney to determine the temporal changes in tissue PO2 during the subacute phase of IRI in conscious rats. In the separate protocol, rats were anesthetized at 24 h or 5 days following reperfusion, to determine the spatial distribution of tissue PO2 and the renal oxygen delivery (DO2) and consumption (VO2). Renal expression of hypoxia-inducible factors (HIF) was determined via western blot analysis. Medullary PO2 measured by telemetry was increased by 23 ± 13% at 24 h and 25 ± 12% at 48 h following reperfusion. Tissue PO2 measured by Clark electrode was consistently greater 24 h after reperfusion than after sham surgery. Tissue PO2 5 days after reperfusion was not significantly different to that at the same time after sham surgery. Renal blood flow and renal DO2 were relatively well maintained in the subacute phase of IRI. In contrast, renal VO2 was 55% less 24 h and 49% less 5 days after reperfusion than after sham surgery. Expression of HIF-1α was considerably less in the cortex, outer and inner medulla of the kidney both at 24 h and 5 days after reperfusion. In contrast, expression of HIF-2α was significantly less in the cortex and outer medulla 5 days after reperfusion than after sham surgery. Renal tissue hypoxia was not detected 24 h and 5 days after ischemia and reperfusion regardless of the methodology. Thus, widespread renal hypoxia does not appear to be obligatory in the subacute phase of renal IRI. The apparent absence of tissue hypoxia may be explained by the marked reduction in renal VO2. In conclusion, at least in this model of IRI, tissue hypoxia is unlikely to drive the progression to CKD.

P13-1Natriuretic Peptide Induces Adipose Tissue Browning and Thermogenesis in Diet Induced Obese MiceHaruka Kimura1),� Tomohisa�Nagoshi1),�Akira�Yoshii1),� Yoshiro�Tanaka1),�Keiichi�Ito1),�Toshikazu�D�Tanaka1),�Michihiro�Yoshimura1)

1）The Jikei University School of Medicine Division of CardiologyIncreasing evidence suggests the possibility that natriuretic peptides induce adipose tissue browning and activate thermogenic program. Using a novel fluorescent thermoprobe, we recently reported that A-type natriuretic peptide (ANP) raises intracellular temperature in cultured adipocytes in a low-temperature sensitive manner. We herein investigated whether ANP exerts a significant impact on adipose tissues in vivo, and assessed the hypothesis that natriuretic peptides induce “browning” of white adipose tissue (WAT) as well as improve brown adipose tissue (BAT) function, leading to thermogenic action using diet induced obese mice. C57BL/6 mice were exposed to high fat diet (HFD) or normal fat diet (NFD) for 13 weeks, and subsequently treated with or without ANP infusion for 3 weeks (0.5 ug/kg/min via osmotic-pump, subcutaneously). Histological analyses revealed that HFD induced dramatically increased BAT cell size with the accumulation of enlarged lipid droplets (whitening, n=3 each). ANP treatment significantly suppressed this whitening response with reduction in lipid droplets size (re-browning, n=3 each). Similarly, HFD induced enlarged inguinal WAT lipid droplets size, which was significantly reduced by ANP treatment. This was associated with a substantial increase in uncoupling protein-1(UCP1) expression in WAT confirmed by immunohistochemical analysis (browning, n=3 each) and UCP1mRNA analysis (NFD+NP 2.7±0.4 fold vs NFD, P<0.01; HFD+NP 2.0c0.6 fold vs HFD, P<0.05, n=5 each). To determine the functional significance of these browning effects of ANP, mice were exposed to cold temperature at 4°C for 4 hours and the rectal temperature was measured. The mice treated with ANP were tolerant to cold exposure, which was more salient in HFD with ANP (Decrease in temperature from baseline (°C): NFD+ANP –4.5±1.0, HFD –3.7±0.5, HFD+ANP –3.4±0.5, vs NFD –13.1±4.5, P<0.05 each, n=3 each). In conclusion, ANP induces WAT browning as well as preserves BAT function in obese models, leading to in vivo thermogenesis in response to cold environment. These findings reveal the compensatory thermogenic action of natriuretic peptides when core body temperature fall due to unfavorable hemodynamic conditions in a state of heart failure.

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P13-5Maternal high-fat diet impairs insulin sensitivity by enhancing NLRP3 inflammasome activationNaotoshi Wada 1), � Hiroyuki � Yamada 1), � Shinichiro�Motoyama2),� Makoto� Saburi1),� Takeshi� Sugimoto1),�Noriyuki�Wakana1),�Satoaki�Matoba1)�1）Kyoto Prefectural Univ of Med, 2）Kyoto Yamashiro General Medical Center[BACKGROUND]Maternal high-fat diet (HFD) has been shown to promote the development of insulin resistance in adult offspring; however, the underlying mechanisms remain unclear.[METHOD AND RESULT]Eight-week-old female wild-type mice (C57BL/6) were fed a HFD or normal diet (ND) one week prior to mating, and received during pregnancy and lactation. Eight-week-old male offspring of both groups were fed a HFD for 8 weeks. Offspring of HFD-fed dams (O-HFD) showed significantly impaired insulin sensitivity compared with ND-fed dams (O-ND). There was no difference in body weight, epidydimal white adipose tissue (eWAT) weight, and cumulative caloric intake between the two groups. However, eWAT adipocyte size was significantly increased in O-HFD (21%, P < 0.05), which was accompanied by augmented mRNA expression levels of proinflammatory cytokines such as TNF-α and MCP-1 (98% and 81%, respectively, P < 0.05). Flow cytometric analysis revealed an increased percentage of M1, but not M2, macrophages (79%, P < 0.05). We thus examined the in vitro polarization of bone marrow-derived macrophages (BMDMs) after conventional stimulation with IFN-γ or IL-4; however, there was no significant difference in both M1- and M2-related genes expression. In contrast, palmitate acid-mediated metabolic activation of BMDMs from O-HFD showed a significantly higher concentration of IL-1β in culture supernatants (45%, P < 0.05). Consistent with in vitro findings, O-HFD showed significantly higher serum levels of IL-1β than O-ND (3.7-fold, P < 0.01), and treatment with NLRP3 Inflammasome Inhibitor MCC950 completely abrogated the exaggerated insulin resistance in O-HFD. These findings suggest that BMDMs from O-HFD is more prone to palmitate-induced inflammasome activation in vitro and in vivo. [CONCLUSION]Our findings demonstrate that maternal HFD exaggerates diet-induced insulin resistance in adult offspring through enhancing NLRP3 Inflammasome activation. Developmental programing of macrophages by maternal HFD could be a potential therapeutic target for preventing metabolic disorders in adult offspring.

P13-2Brown adipose tissue dysfunction is involved in the pathologies in failing heartYohko Yoshida1),�Ippei�Shimizu1),�Tohru�Minamino1)

1）Niigata University

Prognosis of severe heart failure is unacceptably high, and it is our urgent task to find therapies for this critical condition. It has been reported that low body temperature predicts poor clinical outcomes in patients with heart failure, however, underlying mechanisms and pathological implications are largely unknown. Brown adipose tissue (BAT) was initially characterized as a heat generating organ, and studies suggest that BAT has crucial roles for the maintenance of systemic metabolic health. Here we show that BAT dysfunction develops in a murine thoracic aortic constriction (TAC) model, and has a causal role for promoting pathologies in failing heart. TAC operation led to a significant reduction both in intraperitoneal and subcutaneous temperature. TUNEL-positive cells significantly increased in BAT during left ventricular (LV)-pressure overload, and in-vitro studies with differentiated brown adipocytes suggested that the chronic activation of adrenergic signaling promotes apoptosis in these cells. Gain of BAT function model, generated with BAT implantation into peritoneal cavity, improved thermogenesis and ameliorated cardiac dysfunction in TAC. In contrast, genetic model of BAT dysfunction promoted cardiac dysfunction. Metabolomic analyses showed that BAT dysfunction led to an increase of oxidized choline that promoted metabolic dysfunction in the failing heart. Electron microscope study showed that oxidized choline induced mitochondrial dysfunction in vitro as well as in vivo settings. Extracellular flux analyzer indicated that oxidized choline suppresses oxidative phosphorylation in mitochondria. We found that dilated cardiomyopathy patients have lower body temperature, and confirmed by metabolomic study that both choline and oxidized choline are increased in circulation. Maintenance of BAT homeostasis and suppression of oxidized choline would become a novel therapeutic target for heart failure.

P13-3Psychological stress accelerates HFD-induced insulin resistance by enhancing NE activity in eWATShinichiro Motoyama1),� Hiroyuki� Yamada1),�Naotoshi�Wada1),�Makoto�Saburi1),�Takeshi�Sugimoto1),�Noriyuki�Wakana1),�Satoaki�Matoba1)�1）Kyoto Prefectural University of MedicineBackground: Psychological stress (PS) has been linked with the development of cardiovascular diseases (CVD) which is closely associated with insulin resistance (IR); however, the causal effect of PS on IR and its underlying mechanisms remain undefined.Methods and Results: Eight-week-old male wild-type mice (C57BL/6) were exposed to the PS by housing with a larger CD-1 mouse in a shared home cage without physical contact for 10 consecutive days followed by high-fat diet (HFD) feeding. Control mice were housed in the same cage without CD-1 mouse. Before HFD feeding, insulin sensitivity and glucose tolerance were normal in the both groups. After 6 weeks of HFD, insulin sensitivity was significantly impaired in stressed mice without difference in body weight, epididymal white adipose tissue (eWAT) weight, and cumulative food intake between the two groups. Adipocyte size and the number of crown-like structure (CLS) in eWAT were markedly increased in stressed mice (16.8% and 200%, respectively, P < 0.05); however, the percentage of classically activated macrophages assessed by flow cytometric analysis was equivalent between the two groups. In contrast, the percentage of Ly-6G/neutrophil elastase (NE) double-positive cells was markedly increased in CLS of stressed mice versus control mice (34.5% vs 13.0%, P < 0.05), accompanied by the augmented NE activity assessed by ex vivo fluorescent imaging of eWAT (51.3%, P < 0.05). Treatment with NE inhibitor completely abrogated the impairment of insulin sensitivity in stressed mice, accompanied by the reduction of eWAT neutrophils accumulation. Further, in vitro NE release from bone marrow neutrophils upon stimulation with formyl peptide receptor 1 agonist was significantly higher in stressed mice by 14.9% (P < 0.05), while the NE amount before stimulation was comparable between the two groups.Conclusions: Our findings show that PS accelerates the development of HFD-induced IR accompanied by the augmented NE activity in eWAT. Modulation of neutrophils kinetics and functional status may represent a potential therapeutic target for PS-associated IR.

P13-4Adrenomedullin plays a compensatory role in obesity-related hypertensionKosai Cho1),�Miku�Oya2),�Yasuaki�Nakagawa2),�Toshio�Nishikimi2),�Koichiro�Kuwahara3),�Takeshi�Kimura2),�Naoto�Minamino4)�1）Department of Primary Care and Emergency Medicine, Kyoto University Graduate School of Medicine, 2）Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 3）Department of Cardiovascular Medicine Shinshu University School of Medicine, 4）National Cerebral and Cardiovascular Center

【Background】Adrenomedullin(AM), known as a potent long-acting vasodilatory peptide, is secreted and expressed in the kidneys, lung, vasculature, and heart, but recent studies have demonstrated that adipocytes also express and secrete AM. However, little is known about the pathophysiological significance of AM secreted by adipose tissue. 【Purpose/Method】To clarify the function of adipocytes-derived AM, we generated adipocyte-specific AM knockout mice by crossing aP2-cre mice with AM-floxed mice (aP2-AM KO). We evaluated the phenotype of the resulting aP2-AM KO. Blood pressure at 8, 20, and 32 weeks of age, the plasma concentration of AM, and humoral factors affecting blood pressure was measured in aP2-AM KO and control mice.【Results】In comparison with control AM floxed mice, adipocyte-specific AM knockout mice tend to show age-dependent increase in systolic blood pressure under normal chow (88.0±2.1 vs 88.8 ±1.8 at 8weeks of age, 94.5 ±0.8 vs 105.6 ±2.1 mmHg at 32weeks of age). Furthermore, aP2-AM KO mice fed a high fat diet (HFD) exhibited higher systolic blood pressure than control HFD-fed mice (104.3 ±2.3 mmHg in AM adipo-cKO vs 116.7 ±1.8 mmHg in control at 32weeks of age; P ＜ 0.01). AM deficiency in adipocytes did not significantly affect body weight, heart rate, and echocardiographic assessment of cardiac function. Interestingly, aP2-AM KO crossed with AM transgenic mice, in which AM is over-secreted by the liver, showed improved hypertensive phenotype under HFD conditions (101.6 ±2.8 mmHg at the age of 32weeks). Plasma AM concentrations in aP2-AM KO were significantly lower than those in control mice in both normal chow (78.2 ±2.2 vs 92.7 ±3.6 pg/ml; P ＜ 0.05) and HFD conditions (115.1 ±2.9 vs 158.9 ±10.1 pg/ml; P ＜ 0.01). On the other hand, plasma renin activity, aldosterone concentration and urinary catecholamines showed no significant difference between aP2-AM KO and control mice. 【Conclusion】Thus, we conclude that adipocytes produce a substantial amount of AM and that adipocyte-derived AM has a compensatory role in obesity-related hypertension.


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P13-6Loss of periostin ameliorates adipose t issue inflammation and fibrosis in vivoTakahiro Horie1),�Fumiko�Nakazeki1),�Takeshi�Kimura1),�Koh�Ono1)

1）Department of Cardiovascular Medicine, Kyoto University Hospital

Recent evidence suggests that the accumulation of macrophages as a result of obesity-induced adipose tissue hypoxia is crucial for the regulation of tissue fibrosis, but the molecular mechanisms underlying adipose tissue fibrosis are still unknown.In this study, we revealed that periostin (Postn) is produced at extraordinary levels by adipose tissue after feeding with a high-fat diet (HFD). Postn was secreted at least from macrophages in visceral adipose tissue during the development of obesity, possibly due to hypoxia. Postn−/− mice had lower levels of crown-like structure formation and fibrosis in adipose tissue and were protected from liver steatosis. These mice also showed amelioration in systemic insulin resistance compared with HFD-fed WT littermates. Mice deficient in Postn in their hematopoietic compartment also had lower levels of inflammation in adipose tissue, in parallel with a reduction in ectopic lipid accumulation compared with the controls.Our data indicated that the regulation of Postn in visceral fat could be beneficial for the maintenance of healthy adipose tissue in obesity.

P13-7Effects of Tofogliflozin on Cardiac Hypertrophy in Metabolic Model RatsTomonari Kimura1),�Kazufumi�Nakamura1)

1）Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama, Japan.Background: Hypertension and diabetes mellitus (DM) lead to cardiac hypertrophy and heart failure. A sodium-glucose co-transporter 2 (SGLT-2) inhibitor is a novel drug for DM that suppresses reabsorption of sodium and glucose in the proximal tubles and clinically might prevent heart failure. We examined the effect of tofogliflozin, an SGLT-2 inhibitor, on cardiac function in metabolic model rats. Purpose: This study aims to estimate the effect of SGLT2 inhibitor on cardiac hypertrophy, fibrosis, and metabolic state. Methods: We used 4 different groups of male Dahl salt-sensitive (DS) rats and salt-resistant (DR) rats at the age of 6 weeks. All of the rats were fed a high-salt (8% NaCl) and high-fat diet (29.4% kcal from fat) and treated or not treated for 9 weeks as follows: DR rats not treated (n=7), DR rats treated with tofogliflozin (0.005% dietary) (n=7), DS rats not treated (n=13) and DS rats treated with tofogliflozin (n=13). At the end of the protocol, myocardial fibrosis and mRNA expression in left LV tissue were assessed. Results: Urinary glucose concentration was increased in all of the rats treated with tofogliflozin. Tofogliflozin showed hypotensive effect in DS rats (184±1 vs 180±1 mmHg, P<0.05). Left ventricular (LV) hypertrophy was reduced in both DR rats (heart weight/body weight (mg/g), 3.45±0.07 vs 3.25±0.05, P < 0.05) and DS rats (4.43±0.11 vs 4.06±0.06, P < 0.01). Cardiac perivascular fibrosis was reduced in DS rats compared with those in the untreated groups. The expression levels of mRNAs for Nppa, Nppb, Il6, Tgfb1 and Col4a1, and enzymes associated with ketone oxidation (3-hydroxybutyrate dehydrogenase-1 and succinyl-CoA:3-ketoacid CoA transferase) in LV tissue were also reduced in the DS rats treated with tofogliflozin compared with those in untreated rats. Metabolome analysis showed reduction of 3-hydroxybutyrate, a ketone body, in the DS rats compared with those not treated with tofogliflozin. Conclusion: In our study, tofogliflozin ameliorated cardiac hypertrophy and fibrosis. The expression of mRNA for enzymes associated with ketone oxidation is reduced in DS rats treated with tofogliflozin. These results indicate that SGLT2 inhibitor suppresses cardiac hypertrophy and possibly changes cardiac metabolic state in the myocardium.

P13-8Insufficient neonatal ketogenesis triggers the susceptible trait for hepatic steatosisYuichiro Arima1),�Toshifumi�Ishida1),�Koichi�Nishiyama2),�Taishi�Nakamura1),�Satoshi�Araki1),�Koichi�Kaikita1),�Kenichi�Tsujita1)

1）Department of Cardiovascular Medicine, Kumamoto University, 2）International Research Center for Medical Science, Kumamoto UniversityBackground: Many epidemiological reports showed low birth weight babies are at risk of non-communicable diseases in adult. This concept is regarded as the developmental origins of health and diseases (DOHaD). Several researches revealed low birth weight mice showed susceptible trait for high fat diet induced hepatic steatosis in adult. However, its etiologies are almost unknow. Methods: We generated murine low birth weight neonates using caloric restriction during mid-pregnancy. Delivered neonates were classified into low-birth weight group (LBW) and control (Ctrl) group. We harvested livers and Oil-Red staining, RT-PCR analysis and microarray analysis were performed. We also generated knock-out mice of HMG-CoA synthase 2 (Hmgcs2), rate-limiting enzyme for ketogenesis using CRISPR/Cas9 system. Hmgcs2 KO and wild-type mice were analyzed using metabolome analysis and electron microscopic observation. Results: Caloric restriction to pregnant mice produced low birth weight neonates (Birthweight:, LBW 1.47±0.01, n=421, Ctrl 1.86±0.01 n=556, p<0.0001). Oil-red staining revealed higher fat deposition in LBW neonates (LBW 33.3±6.72%, n=6, Ctrl 13.0±3.03%, n=9, p<0.05). Furthermore, ketogenic capacity was impaired in LBW neonates (beta-hydroxybutyrate LBW 0.45±0.07mmol/L, n=11, Ctrl 0.71±0.06 mmol/L, n=14, p<0.05)and rate-limiting enzyme, Hmgcs2 transcription was also decreased. We focused on the neonatal ketogenesis and its relation to the hepatic steatosis and generated HMGCS2 knockout mice using CRISPR/Cas9 system. We generated 10bp and 14bp deletion mutants, both homozygotes showed decreased ketone body concentration and protein expression was also disappeared. Furthermore, HMGCS2 KO neonate showed severe hepatic steatosis. Transmission electron microscopic analysis revealed decreased number of mitochondria and altered morphology. Metabolome analysis also revealed suppressed glycolytic pathway and accumulation of acetyl-CoA. From these data, insufficient ketogenesis caused accumulated Acetyl-CoA, mitochondrial capacity was also decreased. Conclusions: These data represented neonatal impaired ketogenesis caused non-alcoholic fatty liver disease trait through impaired lipid metabolism and mitochondria dysfunction.

P14-1SOX17 mutations in Japanese patients with pulmonary arterial hypertensionTakahiro Hiraide1),�Masaharu�Kataoka1),�Shinobu�Gamou1),�Keiichi�Fukuda1)�1）Keio University School of Medicine

Pulmonary arterial hypertension (PAH) is a rare but serious disease with a poor prognosis.Bone morphogenetic protein type 2 receptor (BMPR2) gene is known as a strong pathogenic factor for PAH. However, BMPR2 mutation accounts for only approximately 20% in sporadic cases of idiopathic PAH. Recently, the rare causal heterozygous variants in SOX17 were significantly over-represented in PAH patients in the cohort of European countries. The purpose of our study is to identify the prevalence and clinical features of idiopathic PAH patients with SOX17 variant in Japan.As a collaborative team from Kyorin University and Keio University in Japan, we performed whole-exome sequencing in 12 Japanese PAH patients and 12 asymptomatic family members in 6 families, as well as in 128 Japanese idiopathic or heritable PAH index cases. Importantly, the study population is a racially, ethnically, and socially homogeneous population. We identified 4 PAH patients and 1 asymptomatic family member with SOX17 mutations. Whole-exome sequencing was negative for mutations in known genes pathogenic for PAH (BMPR2, ACVRL1, ENG, CAV-1, KCNK3, EIF2AK4, and SMADs). Notably, some variants in the presented study and previous study were located within the high-mobility group domain in SOX17, suggesting that mutations within the HMG domain of SOX17 are causative for PAH. In conclusion, the present study reports on 4 Japanese PAH patients with SOX17 mutations, and the findings support SOX17 as a novel causative gene of PAH. Full elucidation of SOX17-mediated pathogenic mechanisms in PAH requires persistent efforts to achieve precision or individualized medicine as a therapeutic strategy for PAH.

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P14-5INHBA is a Novel Gene that Regulates Pulmonary Arterial Hypertension DevelopmentGusty Rizky Teguh Ryanto1),�Koji�Ikeda2),�Kazuya�Miyagawa2),�Keiko�Yagi2),�Yoko�Suzuki1,2),�Ken-ichi�Hirata1),�Noriaki�Emoto1,2)

1）Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 2）Department of Clinical Pharmacy, Kobe Pharmaceutical UniversityBackground Pulmonary arterial hypertension (PAH) is a pulmonary vasculature disease that is pathologically marked by arterial remodeling, especially in the microvessels. This remodeling is known to mainly be caused by the dysfunction of endothelial cell (EC), although the molecular mechanisms surrounding it remain elusive. In this study, we aimed at identifying and elucidating the role of a novel gene in the regulation of PAH development. Methods We searched for genes that regulate lung microvessel homeostasis and determine target gene through DNA microarray analysis using RNAs of human ECs isolated from various vascular beds including lung microvessels, and various human organs including the lung. Once determined, we analyzed the candidate gene expression in lung tissue and pulmonary artery ECs exposed to hypoxia. Next, we analyzed its effect on EC functions via in vitro angiogenesis assays utilizing human pulmonary artery ECs (hPAEC) overexpressing the target gene via retrovirus-mediated transfection. We also generated mice overexpressing the target gene specifically in ECs using VE-cadherin promoter, and exposed them to normoxia or hypoxia (10% O¬2 for 3 weeks) to evaluate its PAH phenotypes. Results INHBA was determined as a candidate gene through DNA microarray analysis, and we confirmed its high and preferential expression in the lung microvessel ECs. INHBA expression was found to be significantly downregulated both in whole lung and pulmonary artery ECs after hypoxia treatment. Interestingly, INHBA overexpression in hPAECs caused a dramatic reduction of EC angiogenic capacities, shown by reduced endothelial tube formation and migration capability with increased apoptosis. Mice overexpressing INHBA in ECs (VEcad-INHBA-Tg) showed significantly higher right ventricular systolic pressure and right ventricular hypertrophy, with evidences of arterial remodeling. Three weeks of chronic hypoxia exacerbated these phenotypes in TG mice compared to control wild-type mice. Conclusion Our data showed that INHBA is an important regulator in PAH development, and thus INHBA is a promising and novel therapeutic target in PAH treatment.

P14-2Pristane might be useful for creating a novel model of CTD-PAH in miceHiroyoshi Mori1),�Tadakatsu�Inagaki1),�Tomohiko�Ishibashi1),�Makoto�Okazawa1),� Takeshi�Masaki1),� Ryotaro�Asano1),�Yoshikazu�Nakaoka1)

1）National Cerebral and Cardiovascular Center Research InstituteOBJECTIVE: Connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH) belongs to group 1 PAH and is clearly d i s t inc t f rom id i opa th i c/hered i ta ry PAH in tha t i t i s pathophysiologically accompanied by strong inflammation and fibrosis in the lungs. However, the authentic murine PAH models, such as hypoxia-induced PH (HPH) model and Su5416/Hypoxia-induced PH (SuHx) model, do not actually reflect the above pathological features of CTD-PAH. Therefore, we aimed to create a novel mouse model of PAH that mimics the pathological characters of CTD-PAH.METHODS: Female C57BL/6 mice (8-week-old) were divided into three groups: an age-matched control group which was exposed to room air in normal oxygen concentration, and the other two groups, which were exposed to 4-week-hypoxia (10%) after administration of pristane (PriHx group) or vehicle (Hx group) for 4 weeks. Right ventricular systolic pressure (RVSP), right ventricle (RV)/left ventricle plus septum (RV/LV+S) weight ratio, and medial wall thickness in distal pulmonary arteries were evaluated. Gene expression analysis in the lung tissues focusing on inflammation and fibrosis was performed by quantitative RT-PCR. Immunohistological analyses with anti-CD45 and anti-CD44 antibodies were also performed.RESULTS: Compared to the Hx group, all the indices of pulmonary hypertension such as RVSP, RV/LV+S ratio, and medial wall thickness were significantly elevated in the PriHx group. In addition, excessive infiltration of inflammatory cells was observed in the lungs of the PriHx group mice. Intriguingly, aggregated cells including macrophages positive for iron-staining were exclusively observed in the lung tissues of the PriHx group. Moreover, most of the above aggregated cells were positive for both CD45 and CD44. Inflammation-related genes, such as I16, Tnfa and Cxcl2, and fibrosis-related genes, such as Col1a1 and Fn1, were significantly upregulated in the PriHx group than in the Hx group.CONCLUSION: These findings indicate that pristane administration combined with hypoxia could become a novel murine PAH model with exaggerated inflammation and fibrosis distinct from HPH model. This model might be useful for elucidating the pathogenesis of CTD-PAH and examining the effect of various drugs on CTD-PAH.

P14-3TrkB might have a role for r ight ventr icular homeostasis under chronic hypoxia-induced PHMakoto Okazawa1),� Tadakatsu� Inagaki1),� Tomohiko�Ishibashi1),�Takeshi�Masaki1),�Hiroyoshi�Mori1),� Ryotaro�Asano1),�Yoshikazu�Nakaoka1)

1）National Cerebral and Cardiovascular Center

Background: Brain-derived neurotrophic factor (BDNF)-Neurotrophic tyrosine receptor kinase type 2 (TrkB) signal transduction pathway plays pivotal roles in various physiological and pathological events. Some reports suggest that up-regulation of TrkB is involved in medial wall thickening of remodeled pulmonary arteries. However, it is not well understood the role of TrkB signals in pathological processes of pulmonary arterial hypertension (PAH). Methods and Results: Quantitative RT-PCR and Western blot were performed to evaluate the expression TrkB expression. When mice were exposed to hypoxia (10 % oxygen), the expression of TrkB mRNA and protein in the lung was increased. To test whether TrkB is involved in hypoxia-induced PH pathogenesis, we conditionally ablated TrkB in mice using the Cre-loxP system. Mice were exposed to room air in normal oxygen concentration or hypoxia for 4 weeks and were subjected to the physiological and histological analyses including right ventricular systolic pressure (RVSP), right ventricle (RV)/left ventricle plus septum weight ratio (Fulton’s index) and medial wall thickness in the distal pulmonary arteries. Global TrkB ablation did not significantly exacerbate RVSP after hypoxia exposure. However, TrkB ablation led to the increase of Fulton’s index with RV chamber dilatation and worsened fibrosis of RV. Conclusion: These findings suggest that TrkB might have a role for cardiac homeostasis under the PAH condition.

P14-4Hypoxia Causes Epigenetic Changes in SMCs of PAH with BMPR2 MutationKazufumi Nakamura1),� Satoshi�Akagi1),� Yukihiro� Saito1),�Kentaro�Ejiri1),�Toru�Miyoshi1),�Masashi�Yoshida1),�Hiroshi�Ito1)�1）Okayama UniversityBackground: Mutations of the bone morphogenetic protein receptor type II (BMPR2) gene are implicated in the pathogenesis of both heritable and idiopathic pulmonary arterial hypertension (PAH). Because mutant alleles are typically of low penetrance, multiple hits by genetic and epigenetic factors are required for disease onset and development in BMPR2 mutation carrier. However, factors of multiple hits remain incompletely understood. The aim of this study was to determine the role of hypoxia in the disease development of PAH with BMPR2 mutation using reprogramming of pulmonary artery smooth muscle cells (PASMCs).Methods and Results: PASMCs were obtained from a patient with heritable PAH with BMPR2 mutation (p.Arg491Gln) who underwent lung transplantation. To clarify the epigenetic modification, we reprogrammed her PASMCs and generated human induced pluripotent stem cells (hiPSCs). We then differentiated the hiPSCs into SMCs again. Hypoxia enhanced PDGF-BB-stimulated proliferation of hiPSC-derived SMCs (hiPSC-SMCs). DNA microarray analysis revealed that expression levels of 14865 genes and 13704 genes were up-regulated and down-regulated, respectively, in PAH-PASMCs compared with those in hiPSC-SMCs. In the up-regulated genes, expression levels of 8040 genes (54%) in hiPSC-SMCs were increased under a hypoxic condition (under 2% 02 for 72 hours). The up-regulated genes included ERK1, AKT2/3, RhoA, HIF1A/3A, IL6 and PDGFRB. In the down-regulated genes, expression levels of 8917 genes (65%) in hiPSC-SMCs were decreased under a hypoxic condition. The down-regulated genes included histone deacetylase 1 and 2 (HDAC1/2), genes related to epigenetic process. Quantitative PCR analysis also revealed that hypoxic condition significantly decreased the expression levels of HDAC1/2.Conclusions: Hypoxia caused enhancement of proliferation and regulation of numerous gene expression including HDAC in SMCs of PAH with BMPR2 mutation.


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P14-6Role of endothelin-1 in the regulation of ALK-1 in human pulmonary arterial endothelial cellsRyo Kawamata1),�Koichi�Sugimoto1),�Yusuke�Kimishima1),�Tomofumi�Misaka1),� Kazuhiko�Nakazato1),� Takafumi�Ishida1),�Yasuchika�Takeishi1)

1）Fukushima Medical UniversityBackground: Pulmonary arterial hypertension entails increases in pulmonary vascular resistance and arterial pressure resulting from vascular lumen narrowing. The abnormal proliferation of pulmonary arterial endothelial or smooth muscle cells as well as the contraction of pulmonary blood vessel can result in pulmonary arterial remodeling leading to right heart failure. Activin receptor-like kinase-1 (ALK-1) is one of the transforming growth factor-β (TGF-β) receptors, which is specifically expressed in endothelial cells. The aberrant functioning of this receptor is implicated in the abnormal proliferation of pulmonary arterial endothelial cells. Moreover, endothelin-1 (ET-1) is known to activate the small GTP protein RhoA, and causes pulmonary blood vessel contraction. Additionally, endothelin receptors are G-protein-coupled receptors, and we have previously demonstrated that Gi, which is a heterotrimeric G protein, functions upstream of RhoA activation. However, the effect of ET-1 on ALK-1 expression and involvement of Gi in pulmonary endothelial cells have not been rigorously examined. The purpose of this study was to investigate the effect of ET-1 on ALK-1 expression in pulmonary arterial endothelial cells and to delineate the underlying molecular mechanisms. Methods: ET-1 was added to the supernatant of cultured human pulmonary arterial endothelial cells, and cellular ALK-1 expression in the cells was observed 18 hours later by Western blot. β-actin was used as an endogenous control. Pertussis toxin (PTX) and Y27632 were used as inhibitors of Gi and Rho kinase, respectively. Results: ET-1 enhanced ALK-1 expression in pulmonary arterial endothelial cells between 0.01 nM and 10 nM. Upregulation of ALK-1 expression by ET-1 was suppressed by the pre-administration of Y27632. In addition, when pulmonary arterial endothelial cells were pretreated with PTX overnight before stimulation with ET-1, ALK-1 expression was suppressed. Conclusion: This study demonstrated that ET-1 increases ALK-1 expression in pulmonary arterial endothelial cells via a pathway that involves Gi and RhoA.

P14-7Expressional analysis of Endothelin-2 in Lung under Hypoxic ConditionDonytra Arby Wardhana1),� Yoko�Suzuki1,2),�Koji� Ikeda1),�Kazuya�Miyagawa1),�CheMyong�Ko3),�Masashi�Yanagisawa4),�Noriaki�Emoto1,2)�1）Clinical Pharmacy, Kobe Pharmaceutical University, 2）Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan, 3）Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, 4）International Institute for Integrative Sleep Medicine, University of Tsukuba, Tsukuba, JapanPulmonary hypertension (PH) is a rare but severe disease that could rapidly progress to the right ventricular failure, leading to the poor prognosis. Detailed molecular mechanisms underlying the pathogenesis of PH remain unclear; however, endothelin-1 (ET-1) has been well recognized to play causative roles in PH. The identification of Endothelin-1 has led to the development of endothelin receptor antagonists, and their clinical benefits for the treatment of PH has been established. In contrast to ET-1, a role of ET-2, which differs from ET-1 only by 2 amino acids and has the same affinity to both ETA and ETB receptor, in the development of PH remain unexplored. In order to analyze the ET-2 expression in lung tissue, we prepared ET2-iCre-Tg mice in which iCre expression was driven by the ET-2 promoter. Subsequently, ET2-iCre-Tg mice were crossed with reporter mice. We also induced PH by exposing mice to chronic hypoxia (10% O2 concentration) using ET-2 hetero knockout (+/-) and the littermate control mice to investigate the role of ET-2 in PH. In addition, we analyzed ET-2 expression in A549 cells, adenocarcinoma human alveolar basal epithelial cells; and BEAS-2B cells, human airway epithelial cells, under normoxic and hypoxic conditions in vitro. We first identified that short exposure (20 minutes) to hypoxia provoked a transient but robust increase in ET-2 mRNA expression in mouse lung. Histological analysis in lung tissue of ET2-iCre-reporter mice exhibited an ET-2 expression in bronchial and alveolar epithelium under normoxic condition. Of note, ET-2 was upregulated in lung endothelial cell under hypoxic condition. Also, ET-2 expression was significantly increased by hypoxia in both A549 cell and BEAS-2B cells. Furthermore, ET-2 +/- mice showed lower right ventricular systolic pressure, Fulton index, and pulmonary artery wall thickness than those in the WT littermates in hypoxia-induced PH model.This study demonstrated the transient activation of ET-2 in the lung at the early time point after hypoxia-exposure and revealed the potential role of ET-2 to modulate the development of PH.

P14-8Loss of Fam13a exacerbates the vascular remodeling in pulmonary artery hypertension.Yuko Kuribayashi1),�Koji�Ikeda1),�Kazuya�Miyagawa1),�Keiko�Yagi1),�Ken-ichi�Hirata2),�Noriaki�Emoto1,2)

1）Laboratory of Clinical Pharmaceutical Science, Kobe Pharmaceutical University, Kobe, Japan, 2）Division of Cardiovascular Medicine, Department of International Medicine, Kobe University Graduate School of Medicine, Kobe, JapanBackground: Pulmonary artery hypertension (PAH), a rare but fetal disease, presents pathological abnormality in pulmonary vessels, called vascular remodeling. In the lungs of PAH patients, the dysfunction of pulmonary artery endothelial cells and pathological proliferation of pulmonary artery smooth muscle cells occur, leading to increased pulmonary vessel resistance. Recently, PAH is hypothesized to be triggered by multi-hits such as environment, heredity, and epigenetics; however, the detailed molecular mechanisms underlying PAH remain to be elucidated. In this study, we focused on family with sequence similarity 13, member A (Fam13a), and explored a potential role of Fam13a in the pathogenesis of PAH. Genome–wide association study revealed a significant association of Fam13a locus with chronic obstructive lung disease and idiopathic pulmonary fibrosis. Methods: We have generated Fam13a-deficient (Fam13a-/-) mice in which LacZ was inserted at downstream of Fam13a promoter, and exposed them to chronic hypoxic condition (10% O2 for 3 weeks) to induce PAH. We measured right ventricular systolic pressure (RVSP), followed by measurement of the Fulton index to analyze pulmonary artery pressure and right ventricular hypertrophy, respectively. Subsequently, the lung was harvested for western blot, real time PCR, and histological analysis. Results and Discussion: Histological analysis using LacZ-staining revealed that Fam13a is expressed in bronchial epithelial cells, and pulmonary artery smooth muscle and endothelial cells. Fam13a-/- mice exposed to chronic hypoxia showed increased RVSP and exacerbated Fulton index compared to those in wild-type mice, indicating that PAH was deteriorated by the loss of Fam13a. We next conducted histological analysis and found that vascular remodeling was accelerated in Fam13a-/- mice. These data strongly suggest that Fam13a in lung vasculature plays a protective role in the development of pulmonary vascular remodeling associated with PAH. Our data shed light on Fam13a as an attractive pharmacotherapeutic target for the treatment of PAH, though detailed molecular mechanisms remain to be elucidated. Keywords Pulmonary Artery Hypertension, FAM13A, Vascular Remodeling, Pulmonary Artery Smooth Muscle Cells

P15-1The role of ORAIP for the coronary artery plaque instability in Familial HypercholesterolemiaHidekimi Nomura1),�Kayoko�Sato1),�Haruki�Sekiguchi1),�Eri�Yamamoto1),�Takuro�Abe1),�Yoshinori�Seko2),�Nobuhisa�Hagiwara�Nobuhisa�Hagiwara1)

1）Tokyo Women's Medical University, 2）Juntendo University Graduate School of MedicineIntroduction: Oxidative stress plays a critical role in the pathogenesis of various disorders including ischemia / reperfusion injury, atherosclerosis, and dyslipidemia. It causes cell damage that leads to apoptosis and atherosclerosis development. The myocardial ischemia / reperfusion induced the high plasma post-translationally-modified secreted-form of eIF5A as Oxidative stress-Responsive Apoptosis-Inducing Protein (ORAIP) levels. On the other hand, heterozygous familial hypercholesterolemia (heFH) is one of the most common genetic diseases and the leading cause of premature cardiovascular disease (CVD). In this study, we investigated whether ORAIP has a role of the atherosclerosis development in the patients with heFH.Hypothesis: The ORAIP / ORAIP-Receptor pathways contribute to the atherosclerotic development and plaque instability by oxidative stress in the familial hypercholesterolemia.Methods and Results: We analyzed plasma ORAIP levels in 60 heFH (60% male, 57.0 ± 13.6 years) and compared as 20 dyslipidemia patients (DL) with LDL-C more than 140 mg/dl (men 85%, age 64.1 ± 13.3 years). The plasma ORAIP levels (normal control range < 10.0 ng/ml) in heFH were significantly elevated compared with those of DL (73.5 ± 46.0 ng/ml vs 47.6 ± 20.3 ng/mL, P<0.05). In addition, plasma oxidized LDL levels were elevated in heFH (156.8 ± 5.2 mg/dL vs 118.2 ± 50.3 mg/dl, P < 0.05) and correlated with plasma ORAIP levels (R = 0.4273, P < 0.05). Furthermore, the immunostaining of the coronary artery obtained from a heFH patients underwent coronary artery bypass graft (CABG) revealed that the co-localization of ORAIP with oxLDL and VSMCs in the atherosclerotic plaque lesion. Finally, we found ORAIP positive VSMCs were TUNEL positive apoptotic cells.Conclusions: Our findings strongly suggest that elevated levels of circulating ORAIP plays a pivotal role in the oxidative stress induced atherosclerotic development and plaque instability in heFH. The elimination of plasma ORAIP with a neutralizing antibody against ORAIP might protect against the plaque rupture and premature CVD.

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P15-5ChGn-2 Deletion Reduces Lipoprotein Retention and PDGF-mediated Aortic Smooth Muscle Cells MigrationImam Manggalya Adhikara1),�Keiko�Yagi2),�Yoko�Suzuki1),�Aristi�Intan�Soraya1),�Koji�Ikeda2),�Ken-Ichi�Hirata1),�Noriaki�Emoto1,2)

1）Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 2）Department of Clinical Pharmacy, Kobe Pharmaceutical UniversityGlycosaminoglycan (GAG) plays an integral part on LDL retention in vascular intimal layer and has emerged as an attractive therapeutic target for atherosclerosis. GAG biosynthesis involves cooperation of numerous enzymes and Chondroitin Sulfate N-acetylgalactosaminyl-transferase-2 (ChGn-2) is one of vital golgi transferase that participates on enzymatic elongation of GAG. ChGn-2 association in atherosclerosis has been documented in our previous work as ChGn-2 expression in mice aorta was upregulated during plaque formation. In addition to being structural protein, GAGs also perform active roles in various signaling pathway and affecting intracellular phosphorylation. However, further investigation is necessary to elucidate ChGn-2 impact on LDL retention and its subsequent signaling properties. In this present study we investigated ChGn-2 crucial contribution to atherosclerotic plaque formation, initial LDL retention, and smooth muscle cell (SMC) phenotype changes. Partial carotid artery ligation was performed on ChGn-2/LDLr null mice to induce diffuse intimal thickening (DIT) and aortic SMCs were isolated to determine cellular binding and signal transduction effect of ChGn-2 gene inactivation. Human coronary artery section revealed that ChGn-2 was expressed in early and advanced atherosclerotic lesion. Mice study demonstrates that ChGn-2 deletion significantly diminishes LDL retention on artificial DIT. Furthermore, LDL accumulation was dramatically reduced after incorporating rhodamine-labeled LDL both in vivo and in vitro, thereby illustrates ChGn-2 essential role on LDL binding machinery. Interestingly, PDGF and PDGFR expression were differentially regulated in mice aorta lacking ChGn-2 after fed with western diet, suggesting a distinct effect on this major signaling pathway that facilitates SMC migration in the advanced stage. To validate these findings, functional assay was conducted on aortic SMC derived from ChGn-2 null mice; and accordingly, it displays abrogation of PDGF-mediated SMC migration via reduced PDGFR phosphorylation, ultimately reveals ChGn-2 modulation on PDGF interaction with its receptor. Our data collectively suggest that ChGn-2 disruption is beneficial throughout atherosclerosis progression thus may serve as a favorable means to treat atherosclerotic-related diseases.

P15-2Phenotypic change of perivascular adipose tissue associated with endovascular damageYusuke Adachi1),� Kazutaka�Ueda1),� Kaoru� Ito2),� Eiki�Takimoto1),�Issei�Komuro1)

1）Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 2）Laboratory for Cardiovascular Disease, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan

Background Perivascular adipose tissue (PVAT), surrounding and directly contacting the adventitia of the vessels, possibly plays a role in the development of atherosclerosis through its direct local action to the vessels. PVAT surrounding carotid and femoral arteries have histological and gene expression characteristics resembling white adipose tissue (WAT) in mice. Recently, it has been attracting much attention that subcutaneous and visceral WAT exert a brown adipose tissue (BAT)-like phenotype under specific conditions such as cold exposure, i.e. browning. On the contrary, browning of PVAT has never been fully elucidated in the context of the development of atherosclerosis.Methods and Results cDNA microarray analyses revealed that endovascular injury upregulates expression of BAT markers including uncoupling protein 1 (UCP1; fold change = 6.23) in mouse carotid artery. The upregulation of UCP1 after injury was confirmed in PVAT by quantitative real-time PCR, western blot and immunohistochemical staining. Of note, an atheroprotective hormone, 17b-estradiol, strongly inhibited the upregulation of BAT marker genes in PVAT after endovascular injury. RNA sequencing using PVAT of injured femoral artery with or without 17b-estradiol revealed that UCP1 is the most down-regulated gene by 17b-estradiol administration (log2-fold change = -6.58, false discovery rate = 0.003).Conclusions We observed PVAT browning after endovascular injury, and this phenomenon may play a substantial role in the development of atherosclerosis. Additionally, 17b-estradiol strongly inhibits PVAT browning, which may provide a novel mechanistic insight into the anti-atheroscrelotic effects of estrogen.

P15-3Adipose-ERK2 Protect From Ectopic Fat Depositions and Vascular Dysfunction in ObesityAyumu Osaki1),�Atsushi�Sato1),�Toyokazu�Kimura1),�Takumi�Toya1),�Yasuo�Ido1),�Takayuki�Namba1),�Takeshi�Adachi1)

1）National Defense Medical CollegeIntroduction: Extracellular signal-regulated kinase (ERK) modulates differentiation and maturation of adipocyte. Adipose tissue plays a critical role in the development of obesity. However, the role of adipose ERK in vivo has not been clarified, yet. This study aims to elucidate the role of the adipose ERK2 in metabolic remodeling and endothelial-dependent relaxation (EDR) in mice model of obesity.Methods and Results: We created adipose-specific ERK2 knock out mice (AE2KO) crossing fatty acid binding protein 4 Cre and ERK2 flox mice and fed them with normal diet (ND) or high-fat and high-sucrose diet (HFHSD) for 24 weeks. HFHSD-fed AE2KO gained higher weight than HFHSD-fed wild-type mice (WT). In HFHSD-fed AE2KO, the weight of subcutaneous fat (SF) was increased and the heterogeneity in sizes of adipocyte was found. Furthermore, the mRNA expression levels of lipoprotein lipase, hormone-sensitive lipase, and proliferator-activated receptor γ (HFHSD-fed WT 5.1±2.1 vs. HFHSD-fed AE2KO 0.47±0.18, P=0.044), which were the master genes of adipocyte differentiation, were markedly decreased in SF. These changes were not seen in visceral fat in spite of the similar reduction of ERK2 expression. The progressive ectopic fat depositions were found in several organs, such as liver, skeletal muscle and perivascular adipose tissue (PVAT) from HFHSD-fed AE2KO. EDR was assessed with acetylcholine (ACh)-induced relaxation in aortic rings with or without PVAT. In ND groups, EDR without PVAT was no difference between WT and AE2KO. PVAT increased EDR in ND-fed WT but failed to increase EDR in ND-fed AE2KO. In HFHSD groups, EDR without PVAT was decreased in HFHSD-fed AE2KO compared with HFHSD-fed WT (ACh 10-6.0 mol/L: HFHSD-fed WT 58±4.5% vs. HFHSD-fed AE2KO 41±2.7%, P=0.018). PVAT decreased EDR in HFHSD-fed WT and mostly eliminated EDR in HFHSD-fed AE2KO. Fluorescence intensity of dihydroethidium stain of PVAT, which indicated adipose superoxide production, was elevated in HFHSD-fed AE2KO.Conclusions: Adipose ERK2 selectively modulated differentiation in SF, suppressed the ectopic fat depositions and protected from organ damages, including hepatosteatitis, mitochondrial dysfunction of skeletal muscle, injured PVAT and endothelial dysfunction with oxidative stress from aorta and PVAT.

P15-4The role of redox regulation of PKG1α on sympathetic nervous system in salt sensitive hypertension.Nobuyuki Tokunaga 1), � Ta ish i � Nakamura 3), � Kenj i�Sakamoto1),�Shokei�Kim-Mitsuyama2),�Kenichi�Tsujita1)

1）Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan., 2）Department of Pharmacology and Molecular Therapeutics, Kumamoto University Graduate School of Medical Sciences, 3）Medical Information Science and Administration Planning, Kumamoto University Hospital, Kumamoto, JapanCyclic GMP and its effector cyclic GMP-dependent protein kinase-1α (PKG1α) transduces NO and/or natriuretic peptide signaling in cardiovascular system. PKG1α oxidation at cysteine42 residues (C42), linking homomonomers by forming a disulfide bond, induces vasorelaxation in resistance arteries. However, the significance of C42 redox sensor in salt sensitivity is unknown. We here compared blood pressure (BP), heart rate (HR), and sodium balance before or after salt loading, using mice harboring either wild-type (WT) or C42S mutant that is redox insensitive PKG1α. First, we conducted a non-reducing SDS PAGE if disulfide dimer formation can be seen in kidney. We found that the disulfide dimer of PKG1α can be observed only in WT kidney subjected with high salt diet or angiotensin II infusion. Consistently with previous reports, we confirmed basal levels of BP in C42S were higher than ones in WT, which is thought to underlie the impairment of vasorelaxation in resistant vessels in C42S. However, even though BP was elevated along with salt loading in WT predominantly during dark period, the further increase was abolished in C42S. High salt diet markedly increased water intake, urinary volume, and urinary sodium excretion, but there were no differences between two genotypes. C42 oxidation in PKG1α thereby led to a decreased slope in pressure-natriuresis relationship, proving PKG oxidation requires a greater increase in BP for sodium excretion. By using telemetry method, we next analyzed Low Frequency/High Frequency ratio (LF/HF) in either heart rate variability or blood pressure variability to evaluate activity levels of sympathetic nervous system in heart or vessels, respectively. In both analyses, the elevation of LF/HF with salt loading significantly occurred during dark period in WT, whereas the nervous activation was certainly prevented in C42S. These results suggest the involvement of sympathetic nervous activation in salt sensitivity is reversible by preventing PKG1α oxidation. C42 redox modulation in PKG1α can be a potent biomarker and therapeutic target for salt sensitive hypertension.


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P15-6Cre expression in hematopoietic cells of smooth muscle cell-targeted Cre recombination miceTomohiko Ishibashi1),� Tadakatsu� Inagaki1),� Hiroyoshi�Mori1),� Takeshi�Masaki1),� Ryotaro� Asano1),�Makoto�Okazawa1),�Yoshikazu�Nakaoka1)

1）National Cerebral and Cardiovascular Center Research InstituteSmooth muscle protein 22 alpha (SM22α)-Cre mouse are widely used as smooth muscle cell (SMC)-specific Cre recombination (SMC-Cre) mice for the study of vascular biology. Accurate knowledge about the specificity of Cre expression is critical for the interpretation of the data obtained by using these mice. It was previously reported that SM22α-Cre mice showed Cre expression in myeloid lineage hematopoietic cells in peripheral blood, spleen, and peritoneum (Shen Z, et al. Biochem Biophys Res Commun. 2012;422:639). However detailed analyses about Cre recombination in hematopoietic lineage cells of SMC-Cre mice are lacking.In this study, we analyzed Cre recombination in hematopoietic lineage cells of SMC-Cre mice, including bone marrow hematopoietic stem/progenitor cells. SMC-Cre mice were crossed with mice that exhibit tdTomato fluorescence after Cre-mediated recombination (SMC-Cre reporter mice), and hematopoietic tissues (peripheral b l o o d , b o n e ma r r ow , a n d s p l e e n ) we r e a n a l y z e d b y immunofluorescent staining and flow cytometry. Robust tdTomato expression was observed in all the three analyzed hematopoietic tissues of SMC-Cre reporter mice, and these tdTomato-positive cells were confirmed to be CD45-positive hematopoietic lineage cells. Cre recombination was detected in not only differentiated myeloid lineage cells but also in lymphoid lineage cells and undifferentiated hematopoietic stem/progenitor cells. Other muscle-targeted Cre recombination mice such as αMHC-Cre mice (cardiac muscle) and MCK-Cre mice (cardiac and skeletal muscle) didn't show any Cre recombination in hematopoietic lineage cells.These results indicate that SMC-Cre mice shows unexpected Cre recombination in hematopoietic cells. Careful attention should be paid when using SMC-targeted Cre recombination mice for the purpose of SMC-specific knockout and lineage tracing studies.

P15-7SREBF1/MicroRNA-33b axis exhibits potent effect on unstable atherosclerotic plaque formation in vivoTakahiro Horie1),�Tomohito�Nishino1),�Takeshi�Kimura1),�Koh�Ono1)

1）Kyoto University

Objective: Atherosclerosis is a very common disease caused by a variety of metabolic and inflammatory disturbances. MicroRNA (miR)-33a within SREBF2 (Sterol regulatory element-binding factor 2) is a potent target for treatment of atherosclerosis through regulating both aspects, however the involvement of miR-33b within SREBF1 remains largely unknown. Although their host genes difference could lead to functional divergence of miR-33a/b, we cannot dissect the roles of miR-33a/b in vivo due to lack of miR-33b sequences in mice, unlike human. Approach and Results: Here, we analyzed the development of atherosclerosis using miR-33b knock-in humanized mice under Apoe-/- background. MiR-33b is prominent both in human and mice on atheroprone condition. MiR-33b reduced serum HDL-cholesterol levels and systemic reverse cholesterol transport. MiR-33b knock-in macrophages showed less cholesterol efflux capacity and higher inflammatory state via regulating lipid rafts. Thus, miR-33b promotes vulnerable atherosclerotic plaque formation. Furthermore, bone marrow transplantation experiments strengthen proatherogenic roles of macrophage miR-33b. Conclusions: Our data demonstrated critical roles of SREBF1-miR-33b axis on both lipid profiles and macrophage phenotype remodeling and indicate that miR-33b is a promising target for treating atherosclerosis.

P15-8Unanticipated roles of IKKβ in vascular calcificationNoboru Ashida1),�Isehaq�Al-Huseini1),�Takeshi�Kimura1)

1）Kyoto University Graduate School of Medicine, Department of Cardiovascular MedicineBackground: Vascular calcification was previously considered as an advanced phase of atherosclerosis; however, recent studies have indicated that vascular calcification can appear in different situations from atherosclerosis, such as renal failure or diabetes. Further, it was reported that usage of statins accelerates the progression of vascular calcification. Nevertheless, there has been a lack of mechanistic insight to explain the difference. For example, the roles of nuclear factor-κB, a major regulator of inflammation, in vascular calcification are poorly explored, although its roles in atherosclerosis were well analyzed. Herein, we investigated the roles of nuclear factor-κB signaling in vascular calcification.Methods and Results: We produced mice with deletion of IKKβ, an essential kinase for nuclear factor-κB activation, in vascular smooth muscle cells (VSMCs; KO mice) and subjected them to the CaCl2-induced aorta injury model. Unexpectedly, KO mice showed more calcification of the aorta than their wild-type littermates, despite the former's suppressed nuclear factor-κB activity. Cultured VSMCs from the aorta of KO mice also showed significant calcification in vitro. In the molecular analysis, we found that Runt-related transcription factor 2 (Runx2), a transcriptional factor accelerating bone formation, was upregulated in cultured VSMCs from KO mice, and its regulator β-catenin was more activated with suppressed ubiquitination in KO VSMCs. We also made mice with overexpression of kinase-active IKKβ in VSMCs, and observed opposite in vivo and in vitro results of KO mice. Furthermore, we made mice in which kinase-dead IKKβ was overexpressed in VSMCs on the background of KO mice, and examined VSMCs from them. Surprisingly, we found that kinase-independent function of IKKβ is involved in suppression of calcification via inactivation of β-catenin, which leads to suppression of Runx2 and osteoblast marker genes.Conclusions: IKKβ negatively regulates VSMC calcification through β-catenin– Runx2 signaling in a kinase-independent manner, which revealed a novel function of IKKβ on vascular calcification.

P16-1Atrial Fibrillation Detection Using Convolutional Neural NetworksXue Zhou1),�Xin�Zhu1),�Keijiro�Nakamura2),�Mahito�Noro3)

1）University of Aizu, 2）Ohashi Medical Center, 3）Sakura Medical CenterAtrial fibrillation (AF) is the most common cardiac arrhythmia and draws great attention in clinical practice because of its continuously growing prevalence in aging society. Electrocardiogram (ECG) is the most important and convenient diagnostic tool for heart diseases, but the ECG of patients with heart disease is very complex and needs an experienced cardiologist to interpret correctly. This study aims to develop an automatic AF detection technique using artificial intelligence techniques. The program is trained and validated using MIT-BIH atrial fibrillation database composed of 25 long-term ECG recordings. Each record has a duration of about 10 hours and contains two-lead ECG signals sampled at 250Hz. The whole processing is composed of signal preprocessing and a CNN detector. Preprocessing includes denoising, resampling, segmentation, and normalizing to generate training samples and testing samples. The detector is composed of two CNN layers and two fully-connected layers for automatic AF detection. Totally, we selected 20,056 training samples with 10,028 AF samples and 10,028 Non-AF samples to keep data balance. 3,112 testing samples were selected with 1123 AF and 1989 Non-AF. Training samples are used to train the CNN detector and testing samples are used to evaluate the detection performance. Finally, we get a 98.9% sensitivity, a 99.0% specificity and 99.0% accuracy for this detection system. Training this detection model using 20,056 data needs about 8.7 hours, but testing using 3,112 samples just needs about 12 seconds.To evaluate the reliability of these detectors, we use testing samples with different AF proportions including 10%, 35%, 50%, 75% and 95%. Limited by the number of testing samples, we randomly select 1,000 samples from original testing samples to perform this evaluation. Through testing 20 times for each AF proportion, the accuracy is always larger than 98.6%, and the higher AF proportion, the higher sensitivity almost.The proposed CNN detector demonstrates an excellent performance for AF detection and may assist physicians for AF diagnosis or reduce false alarm for clinical monitoring. In the future, we will optimize the networks and achieve a real-time detection.

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P16-5SERCA2 C674S heterozygote knock-in mice with angiotensin infusion leads to ventricular arrhythmiaKei Ito1),�Hirotaka�Yada1),�Shunpei�Horii1),�Takumi�Toya1),�Yuji�Nagatomo1),�Bonpei�Takase1),�Takeshi�Adachi1)

1）National Defense Medical CollegeBackground: It remains unsolved what the role of sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2) plays in ventricular electrical stability under pathological conditions. Oxidative stress induced in heart failure selectively modified Cys674 on SERCA2. To test the role of Cys674 on SERCA2 in the induction of ventricular arrhythmia (VA) with cardiac hypertrophy, we employed SERCA2 C674S heterozygote knock-in mice (SKI) with angiotensin II- infusion (ANG) to create pathological condition.Methods: Wild type mice (WT: n=14) and SKI mice (n=14) were subjected to continuous subcutaneous infusion of ANG (3.0mg/kg per day) for a week. Other SKI mice (n=13) were pretreated with a ryanodine receptor (RyR) stabilizer, dantrolene (DAN, 20mg/kg/day, i.p.) followed by ANG. We made distilled water infusion groups in either WT (n=11) or SKI (n=11) as control, and electrophysiological studies were performed.Results: ANG elevated blood pressure and represented cardiac hypertrophy with fibrosis similarly both in WT and SKI mice. SKI mice showed the longer QTc interval than WT mice in control. ANG infusion prolonged QTc further, and SKI mice with ANG revealed the most prolonged QTc interval. DAN prevented QT prolongation in SKI with ANG. Under programmed electrical stimulation, SKI mice with ANG had significantly higher incidence of VA (0/11 in WT/SKI control, 0/14 in WT with ANG vs. 8/14 in SKI with ANG, P<0.01). DAN also reduced the incidence of VA in SKI mice with ANG (2/13 in DAN (+) vs. 8/14 in DAN (-), P<0.05). In optical mapping experiment, burst pacing induced ventricular tachycardia, which was originate from outflow tract in SKI mice, suggested that the mechanism of VA in SKI mice could be triggered activity. Conclusions: Partial dysfunction of SERCA2 in SKI was associated with prolonged QTc intervals and higher VA induction rate in cardiac hypertrophy with ANG. Inhibition of abnormal Ca2+ leakage from RyR by DAN prevented QT prolongation and high incident of VA in SKI mice with ANG. Oxidation of Cys674 on SERCA2 under pathological conditions might play an important role in cardiac Ca2+ handing and arrhythmogenesis.

P16-2Non-uniform calcium dynamics of the atrial cells and Purkinje fibers with references to T tubulesHideo Tanaka1),�Taka-aki�Matsuyama1)

1）Kyoto Prefectural University of Medicine

[Background and aim] Efficient pumping of the heart is mediated by the rapid and spatially uniform Ca2+ transients, which are ensured by the transverse tubules (T tubules), i.e., fine invaginating structures of the cell membrane that are extensively distributed in the cardiomyocytes. As compared with ventricular myocytes, atrial cells and Purkinje fibers are relatively poor in distribution of T-tubular networks, especially in small mammals, e.g., the mouse and rat. However, it is unclear whether or how the intracellular Ca2+ ([Ca2+]i) dynamics are altered in these two types of cells in the heart. To address this issue, we visualized [Ca2+]i dynamics of the rat atrial myocytes and mouse Purkinje fibers in perfused hearts with references to T-tubules. [Materials and Methods] Under loading of the heart with Ca2+ indicator fluo-4, [Ca2+]i dynamics of the heart was visualized mesoscopically by macro-zoom fluorescent microscopy and microscopically by rapid scanning confocal microscopy at 100 frames/s in room temperature. Precise T-tubules were identified by confocal microscopic images of a membrane dye, di-4-ANEPPS. [Results] We found that [Ca2+]i dynamics are spatiotemporally non-uniform in atrial myocytes during relatively higher excitation possibly due to the paucity of T-tubules. Higher frequency excitation of the heart exhibits more remarkable non-uniformity in [Ca2+]i dynamics in atria. Purkinje fibers in the infarct border zone also exhibits inhomogeneous [Ca2+]i dynamics even during low-frequency excitation. [Summary and conclusion] Combined analyses of [Ca2+]i dynamics and T-tubular distribution may provide deeper insights into understanding the dysfunctions of the heart, e.g., conductivity and mechanical performance.

P16-3THREE-DIMENSIONAL STRUCTURAL ANALYSIS OF MUTANT RYR2 CHANNELS ASSOCIATED WITH CPVTJingshan Gao1),� Takeru�Makiyama1),� Seiko�Ohno2),� Yuta�Yamamoto1),�Yimin�Wuriyanghai1),�Minoru�Horie3),�Takeshi�Kimura1)

1）Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 2）Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, 3）Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical ScienceIntroduction: Ryanodine receptors (RyRs) are large homotetrameric calcium-permeant ion channels in the membrane of the ER/SR. In cardiac muscle, the Ca2+ influx through the surface membrane during the action potential activates RyR2 channels, a process called Ca2+ induced Ca2+ release. Mutations in the RYR2 gene are associated with catecolaminergic polymorphic ventricular tachycardia (CPVT) which cause lethal ventricular arrhythmias resulting from increased diastolic calcium leak from mutant RyR2 channels. However, the disease causing mechanisms of each mutation are still unclear. In this study, we aimed to analyze the 3D localization of the CPVT-related RYR2 mutations by using an in silico 3D structural model of RyR2.Methods: Using a 3D structural model developed by cryo-electron microscopy (EM) images of RyR2 (PDB: 5go9, 5goa, Peng et al. Science 2016), we mapped 77 RYR2 mutations which we identified in 93 probands with CPVT. Results:Overviewing the 3D structure of RyR2, we found that the mutations are regionally distributed, mainly in three parts known as “hot-spots” regions. In the N-terminal part (residues 1-642), mutations G172E, R196L, R196Q located on the inner loop of the cytoplasmic canopy alter the charges of the amino acids which might lead to the instability of the intersubunit interaction resulting in the malfunction of RyR2 channels. In the periphery part of the cytoplasmic canopy (653-3612), 22 mutations are located near two predicted FKBP12.6 binding sites. In the channel part (3613-4968), we found 12 mutations are around the calcium sensor and 4 mutations are close to the pore-forming segment (especially, the mutant V4821I is just on the segment). Besides, we performed mutagenesis on the RyR2 crystal structure and found that mutations like R420Q and R420W, which change the amino acid polarity might cause conformational instabilities of the RyR2 tetramers, which favor a closed-to-open state transition.Conclusion: Using an EM-derived 3D structural model, we found significant mutations which might induce conformational changes of the RyR2 tetramers. 3D structural analysis is useful to understand the pathophysiological mechanisms of CPVT-associated RYR2 mutations.

P16-4Glucose fluctuations in diabetic mice induce cardiac sinoatrial node dysfunction.Takahiro Oniki1),�Yasushi�Teshima1),�Yumi�Ishii1),�Shintaro�Kira1),�Ichitaro�Abe1),�Kunio�Yufu1),�Naohiko�Takahashi1)

1）Department of Cardiology and Clinical Examination, Faculty of Medicine, Oita University

Backgrounds: Diabetes mellitus (DM) is a major risk factor for cardiovascular diseases such as ischemic heart disease, heart failure, and arrhythmias. For decades, risk of cardiovascular complications in DM has been believed to correlate with elevated glycated hemoglobin and fasting glucose levels. However, several large-scale clinical trials have recently proposed adverse effects caused by intensive glycemic control which may induce large changes of blood glucose level. We previously reported that glucose fluctuations increase the incidence of atrial fibrillation by promoting cardiac fibrosis mediated via increasing reactive oxygen species levels. In the present study, we investigated whether glucose fluctuations may morphologically and functionally damage cardiac sinoatrial node.Methods and Results: Male mice (C57BL/6) were divided into groups. Hyperglycemic animal models were prepared by intraperitoneal injection of streptozotocin (50 mg/kg/day) for 5 days. Glucose fluctuations (GF) were induced by repeating oral administration of glucose, or fasting and free feeding after confirming hyperglycemia. Heart rate was decreased in GF group. The excised hearts were used to evaluate the sinus node function recovery time (SNRT) in the Langendorff perfusion apparatus. The SNRT in GF group was prolonged more. Morphological observation using a transmission electron microscope revealed swelling of mitochondria and obvious atrophy of sinoatrial node.Conclusion: Blood glucose fluctuation caused morphological and functional damage in sinoatrial node in diabetic mice.


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P16-6Inhibition of HDAC 6 manipulates electrophysiological properties of HL-1 cellsPeili Li1),� Yasutaka� Kurata2),� Fikri� Taufiq1),� Yasuaki�Shirayoshi1),�Ichiro�Hisatome1)

1）Tottori University, 2）Department of Physiology, Kanazawa Medical University

Background: The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (IKr) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Histone deacetylase (HDAC) 6 inhibition increases wild-type hERG expression and currents and rescues mutant hERG expressing in HEK293 cells via posttranslational modification. Effects of HDAC6 on the electrophysiological properties of HL-1 mouse cardiomyocytes including endogenous ERG activity remain unknown.Methods and Results: HL-1 cells were treated with a pan-HDAC inhibitor, TSA and a selective HDAC6 inhibitor, TBA for 24 hours. Immunoblot showed that both inhibitors increased ERG expressions. TSA, but not TBA increased the acetylated histone H3 level. Immunochemistry revealed that TBA increased expression of the ERG on the cell surface and in the endoplasmic reticulum. Electrophysiological techniques showed that TBA treatment increased IKr which was complete blocked by E4031. Action potential durations (APD) were shortened by TBA. E-4031 dramatically prolonged APD at 90% repolarization (APD90) in both control (vehicle) and TBA- treated HL-1 cells. There were no significantly differences in APD90 between control and TBA- treated groups in presences of E-4031 (APD90=210±6.7 and 217±8.1ms, respectively. p=0.57) . TBA enhanced hERG expression in cardiomyocytes derived from human ES cells.Conclusion: Inhibition of HDAC enhances ERG protein and the channel function. Manipulating HDAC6 enable to alter electrophysiological properties o f HL-1 . HDAC6 inh ib i t i on regu la tes ERG may be v ia posttranslational modification and provide a potential therapeutic value for LQT2.

P16-7Pacemaker activity generated by CRISPR/Cas9 based genome editing for Kcnj2Kensuke Ihara1),�Tetsuo�Sasano2),�Masahiro�Yamazoe1),�Tetsushi�Furukawa1)

1）Department of Bio-informational Pharmacology, Tokyo Medical and Dental University, 2）Department of Cardiovascular Physiology, Tokyo Medical and Dental UniversityIntroduction: Previous attempts to make biological pacemaker by gene delivery had a limitation that the effect was transient. Application of genome editing by CRISPR/Cas9 is expected to generate permanent gene knockout. The purpose of this study is to generate permanently functional biological pacemaker utilizing CRISPR/Cas9 based genome editing, targeting Kcnj2, a gene coding IK1 channel.Methods and Results: We constructed Cas9 and CRISPR guide RNA sequence for Kcnj2 (CRISPR-Kcnj2), and performed in vitro and in vivo genome editing. Adenovirus harboring CRISPR-Kcnj2 was applied on isolated neonatal mouse cardiomyocytes (NMCs). NMCs infected with CRISPR-Kcnj2 showed a significant increase in the ratio of spontaneous beating cells compared to non-infected NMCs (9.0% vs 2.4%, p<0.05). The adeno-associated viral vector (AAV) harboring CRISPR-Kcnj2 or only Cas9 (control) was directly injected into the mice heart by open chest surgery. Four weeks or 6 months after the injection, the hearts were excised, followed by ex vivo electrophysiological study with optical mapping. We created complete atrioventricular (AV) block by ethanol injection into the AV node. Constant escaped ventricular rhythm during AV block was observed in all CRISPR-Kcnj2 injected mice 4 weeks after injection, whereas none of the control mice showed escaped rhythm (n=3 each). The optical mapping revealed the origin of escaped ventricular rhythm corresponded to the injection site of AAV with CRISPR-Kcnj2. Sequencing of the target region showed the successful in vivo genome editing resulting in the knockout of Kcnj2 gene by frameshift. Six months after injection, although we confirmed in vivo genome editing was maintained, no ventricular escaped rhythm was seen in CRISPR-Kcnj2 injected mice heart during AV block.Conclusion: Genome editing for Kcnj2 with CRISPR/Cas9 can generate pacemaker activity, however, it is difficult to maintain the pacemaker activity in the long term with this approach.

P17-1Mechanisms for Ca2+ signaling defects underlying diastolic dysfunction in diabetic cardiomyopathyYoshinori Mikami1),�Masanori� Ito1),�Shogo�Hamaguchi2),�Shingo�Murakami1,3),�Taichiro�Tomida1）,�Iyuki�Namekata2),�Hikaru�Tanaka2),�Satomi�Adachi-Akahane1）

1）Department of Physiology, Faculty of Medicine, Toho University, 2）Department of Pharmacology, Faculty of Pharmaceutical Sciences, Toho University, 3）Faculty of Science and Engineering, Chuo UniversityDiabetes mellitus (DM) causes cardiomyopathy in diabetic patients and is a dominant risk factor for heart failure. The left ventricular (LV) diastolic dysfunction represents the earliest preclinical manifestation of diabetic cardiomyopathy. The defective Ca2+ signaling is one of the candidate mechanisms of diastolic dysfunction. The aim of this study is to elucidate the underlying molecular mechanism of Ca2+ signaling defects of diabetic cardiomyopathy. DM was induced in mice by streptozotocin (STZ) injection. In the STZ-induced DM (STZ-DM) model mice 4 weeks after STZ injection (STZ-4W), ejection fraction was preserved, but diastolic function was impaired. These cardiovascular pathologies were similar to HFpEF in the early stage of diabetic cardiomyopathy. In the isolated ventricular myocytes from STZ-DM mice, the Ca2+ transient decay rate was slower than that from control mice. Ca2+ uptake into SR via SERCA is accelerated by PKA-dependent phosphorylation of phospholamban (PLN)-Ser16. In the ventricles of STZ-4W mice, the level of PLN phosphorylation was significantly lower than that of control mice. However, the expression levels of βARs, the maximal myocardial response to βAR stimulation or sensitivity to βAR stimulation was not different between STZ-4W and control mice. Next, to elucidate whether insulin signaling is responsible for LV diastolic function, we examined the effects of insulin administration by osmotic pump for 3 weeks from 1 week after STZ injection. The level of PLN-Ser16 phosphorylation and the relaxation rate of the isolated ventricular myocardium were recovered to the control level in the insulin-responder STZ-4W mice. We further confirmed that insulin is required for the maintenance of basal PLN-Ser16 phosphorylation in the primary cultured neonatal mouse ventricular myocytes. These results indicate that the reduction of PLN-Ser16 phosphorylation level caused by the impaired insulin signaling is responsible for LV diastolic dysfunction in the early stage of diabetic cardiomyopathy.

P17-2Physiological role of TRPC6 upregulat ion in hyperglycemic rodent heartsSayaka Oda1,2),� Takuro�Numaga-Tomita1,2),� Akiyuki�Nishimura1,2),�Motohiro�Nishida1,2,3)

1）Division of Cardiocirculatory Signaling, National Institute for Physiological Sciences (Exploratory Research Center on Life and Living Systems), National Institutes of Natural Sciences, 2）Department of Physiological Sciences, SOKENDAI (School of Life Science, The Graduate University for Advanced Studies), 3）Department of Translational Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kyushu UniversityReceptor-activated Ca2+-permeable cation channels (RACCs) have been attracted attention as molecular targets of next generation Ca2+ channel blocker. Transient receptor potential canonical (TRPC) proteins are molecular entities of RACCs and reportedly upregulated in various diseases and pathologies. Especially in the cardiovascular systems, diacylglycerol-activated TRPC channel proteins (TRPC3 and TRPC6) are reportedly upregulated in pathologically hypertrophied hearts. We have recently revealed that upregulation of TRPC3 proteins negatively regulates proteasome-dependent degradation of NADPH oxidase 2 (Nox2), a reactive oxygen species (ROS)-generating enzyme, independently of channel activity, resulting in induction of ROS-dependent cardiac fibrosis (stiffness) and myocardial atrophy in mouse hearts. In contrast, there is no direct causal-relationship between TRPC6 and heart failure, although TRPC6 is known to be highly upregulated in various pathologies including hyperglycemia, ischemia, and hemodynamic loading. Therefore, we investigated the role of TRPC6 upregulation in hyperglycemia-induced risk of heart failure, especially focusing on the crosstalk between TRPC6 and TRPC3-Nox2 protein complex. The abundance of TRPC6 protein was increased while that of Nox2 protein was decreased in streptozotocin (STZ)-treated mouse hearts and neonatal rat cardiomyocytes (NRCMs) treated with high glucose. Treatment of wild type, TRPC3-deleted and TRPC6-deleted mice with STZ showed significant increases in blood glucose level to a similar extent, but only TRPC6-deficient mice showed severe reduction of cardiac contractility and excess production of oxidative stress. TRPC6-silenced NRCMs treated with high glucose showed marked increases in ROS production and expressions of inflammatory cytokines. In TRPC3/TRPC6/Nox2-expressing cells, these three parties formed a ternary complex, and inhibited increase of Nox2 protein expression by TRPC3. TRPC6 channel activity was not required for counteracting TRPC3-Nox2 complex by TRPC6 protein. Upregulation of TRPC6 in cardiomyocytes exposed to hyperglycemic condition inhibits formation of TRPC3-Nox2 complex and suppresses Nox2-dependent ROS signaling. These results suggest that TRPC6 upregulation contributes to adaptation of the heart against hyperglycemia-induced oxidative stress.

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P17-6SGLT1 exhibits ischemic tolerance and contributes to cardioprotection in diet-induced obese miceAkira Yoshii1),�Tomohisa�Nagoshi1),�Yusuke�Kashiwagi1),�Haruka�Kimura1),� Yoshiro�Tanaka1),� Takuya�Yoshino1),�Michihiro�Yoshimura1)

1）The Jikei University School of Medicine, Division of CardiologyAlthough recent large clinical trials showed that SGLT2 inhibitors reduced cardiovascular events in diabetic patients, the regulation of cardiac SGLTs (SGLT1 is the dominant isoform), as compared to that of GLUTs, under pathological conditions remains incompletely understood. Since glucose becomes an important preferential substrate for myocardial metabolism during ischemia-reperfusion injury (IRI) , we hypothesized that SGLT1 contributes to cardioprotection during acute phase of IRI via enhanced glucose utilization, particularly in insulin resistance phenotypes. The hearts from either high fat diet (HFD) for 12 weeks or normal fat diet (NFD) mice were subjected to IRI using Langendorff model. The immunoblotting with plasma membrane fractionations revealed that GLUT4 expression was significantly increased after IRI in NFD, which was substantially attenuated in HFD. In contrast, SGLT1 expression was maintained constant during IRI regardless of diet conditions. SGLT2 was not detected in the heart. To test the functional significance of the ischemic tolerant SGLT1 expression in HFD, hearts were perfused with either non-selective SGLT inhibitor, phlorizin (100µM), or selective SGLT2 inihibitors, tofogliflozin (5µM) or ipragliflozin (10µM). After IRI, HFD significantly impaired left ventricular developed pressure (LVDP) recovery compared with NFD (% recovery of baseline; 21.3±2.3 vs. 68.3±7.1, n=8, p<0.01). This was associated with a significant reduction in myocardial glucose uptake, mainly due to blunted GLUT4 reaction to IRI in HFD. Although phlorizin perfusion impaired LVDP recovery in NFD (29.5±4.5%, n=6, P<0.01), further impaired LVDP recovery (11.5±2.4%, n=9, P<0.01 vs. HFD control) with dramatically increased infarct size (indicated by CPK release into perfusate and TTC staining), as well as reduction in glucose uptake were observed in HFD with phlorizin. On the other hand, SGLT2 inhibitors perfusion did not significantly affect cardiac function during IRI in both diet conditions. In conclusion, cardiac SGLT1 (but not SGLT2) plays a compensatory protective role during acute phase of IRI via enhanced glucose utilization, particularly under insulin resistance condition, in which IRI-induced GLUT4 upregulation is compromised.

P17-3Activation of SERCA2 ameliorated endothelial function and skeletal muscle function of db/db miceToyokazu Kimura1),�Ayumu�Osaki1),� Kei� Ito1),� Takumi�Toyo1),�Yasuo�Ido1),�Hirotaka�Yada1),�Takeshi�Adachi1)

1）National Defense Medical College

Introduction: Sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), which is a membrane protein located at endoplasmic reticulum (ER), has been found to be associated with pathogenesis and cardiovascular complications of type 2 diabetes (T2D). Recently, it was reported that an allosteric SERCA2 activator, CDN1163, ameliorated glucose metabolism and hepatosteatosis in ob/ob mice. However, the effect of CDN1163 for cardiovascular complications of T2D had not been clarified. In this study, we investigated whether CDN1163 ameliorated glucose metabolism and vascular/skeletal muscle dysfunctions using diabetes (db/db) mice. Methods and Results: Either CDN1163 100 mg/kg or vehicle was intraperitoneally injected to 15 week-old-male control (db/+) and db/db mice for 5 consecutive days. CDN1163 significantly decreased blood glucose at 60, 120 minutes in oral glucose tolerance test and also decreased the serum insulin levels at 120 minutes of db/db mice compared with control mice . CDN1163 also amel iorated hepatosteatosis of db/db mice. CDN1163 improved the oxygen consumption mainly from skeletal muscle with db/db mice in the early time of exercise. In isometric tension measurement of aortic rings, in vivo administration of CDN1163 improved acetylcholine- and sodium nitroprusside- induced relaxation and ex vivo administration, when CDN1163 directly added to Krebs buffer, improved acetylcholine-induced relaxation in db/db mice compared with control mice. Conclusions: CDN1163 improved vascular NO bioactivity, skeletal muscle function, hepatosteatosis, and glucose-metabolism in db/db mice. The activation of SERCA2 is an appealing strategy for the cardiovascular complications in T2D.

P17-4Understanding the pathogenesis of diastolic dysfunction: from the whole heart to the myofilamentMark T Waddingham1),�Hirotsugu�Tsuchimochi1),�Takashi�Sonobe1),�Nazha�Hamdani2),�Mikiyasu�Shirai1),�James�T�Pearson1),�Takeshi�Ogo1)

1）National Cerebral and Cardiovascular Center, Suita, Osaka, Japan, 2）Ruhr University Bochum, Bochum, GermanyMyocardial contraction and relaxation is principally governed by the movement of cross-bridges (CBs) of the myosin thick-filament to and from the actin thin-filament, respectively. CB movement is regulated by post-translational modifications (PTMs) of myosin thick-filament and actin-thin filament accessory proteins as well as the sarcomeric protein, titin. Under physiological conditions, myofilament protein PTMs are fine-tuned to regulate myocardial function on a beat-to-beat basis. Although in disease states, such as type-2 diabetes mellitus (T2DM), this process can become disrupted leading to diastolic dysfunction. Here we describe a unique work-flow for assessing global cardiac function, actin-myosin CB dynamics in the in situ beating heart, cardiomyocyte mechanics and PTMs of myofilament and sarcomeric proteins to allow for a comprehensive understanding of the pathogenesis of diastolic dysfunction, using the Goto-Kakizaki (GK) rat, a model of T2DM as an example. Using SPring-8 synchrotron radiation (BL40XU) as a source for small angle x-ray scattering (SAXS), anesthetized rats were positioned with their myocardial fibre direction at an oblique tangent to the x-ray beam direction, for the collection of actin-myosin CB dynamics recordings in the in situ beating heart. Simultaneous cardiac catherization was performed to correlate changes in CB dynamics with global cardiac function. Cardiomyocyte mechanics and sarcomeric/myofilament protein phosphorylation were assessed in myocardial tissue collected from rats after the SAXS protocol. We found that GK rats developed signs of early global LV diastolic dysfunction, in addition to reduced CB extension toward actin in diastole. In isolated, skinned cardiomyocytes from GK rats, increased passive forces were demonstrated, consistent with global diastolic dysfunction. Furthermore, site-specific hypophosphorylation of the sarcomeric protein titin was also observed in the GK rats. Given the important role of titin in modulating both cardiomyocyte stretch and myosin CB extension, we conclude that changes in t it in phosphorylation may underlie the development of diastolic dysfunction in T2DM. We are currently applying this unique approach to understand the pathophysiological and functional changes of the right-ventricular myocardium in pulmonary arterial hypertension.

P17-5Impact of insulin resistance on right heart failure in sugen chronic hypoxia ratsHuiling Jin1),�Takashi�Sonobe1),�Hirotsugu�Tsuchimochi1),�Mikiyasu�Shirai2),�Takeshi�Ogo2),�James�T�Pearson1)

1）Department of Cardiac Physiology National Cerebral Cardiovascular Center Research Institute, 2）Department of Advanced Medical Research in Pulmonary HypertensionPatients with comorbid diabetes are more at risk and display more severe pulmonary dysfunction than non-diabetics. The purpose of this study is to determine the effects of prevailing systemic endothelial dysfunction associated with insulin resistance on pulmonary vascular and ventricular function in insulin resistant Goto-Kakizaki (GK) rats utilizing cardiac pressure-volumetry, echocardiography and high resolution in vivo synchrotron microangiography (vessels 30-400μm). Further, we examined the roles of eNOS and RhoA/ROCK activation in the pathophysiological changes. Six-week old male Wistar and GK rats were treated with sugen 5416 (20mg/kg sc) followed by 3 weeks of chronic hypoxia prior to imaging or functional assessment at 3 or 6 weeks post sugen (n=8/group). Fasudil (100mg/kg/d) or normal drinking water was provided during the final 3 weeks in normoxia in the latter groups. Mortality occurred during chronic hypoxia in GK rats, but not Wistar controls, and in both strains in the following 3 weeks. Three-weeks post sugen GK rats had a higher haematocrit (67±1%) and Fuller ratio (0.58±0.01), but lower systolic RV pressure (74.2± 2.8mm Hg) in comparison to Wistar rats (47.3±3.2%, 0.51±0.02 and 93.2±4.4 mm Hg, n=4-8). At 6 weeks, RV pressure was further elevated in Wistar rats only (128.0±6.3 vs 73.3±4.6 mm Hg). Myocardial infarctions were present in the RV and septum in GK rats only (both 3 and 6 weeks post sugen, >90%). Right coronary and pulmonary endothelial dysfunction was present in both strains from 3 weeks, but was exacerbated in the lungs in GK rats at 6 weeks. Acute and chronic fasudil treatment ameliorated pulmonary constriction in both strains, and elevated RV pressure in chronically treated GK rats (133±1.0 vs GK 104±10.5 mm Hg). Taken together, endothelial dysfunction was more pronounced in the right coronary and pulmonary circulations in GK rats compared to Wistar at 3 and 6 weeks respectively. ROCK activation contributed to reduced RV pressure development in insulin resistant rats as this limitation was removed by chronic fasudil treatment. RV hypertrophy was accelerated, and thrombosis consistently occurred in the right heart when pulmonary hypertension developed in combination with insulin resistance.


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P17-7Glucagon plays protective role against catecholamine surge in heart failureKazuyuki Nishimura1),� Yasuko� K.� Bando1),� Takahiro�Kamihara1),� Yasheng�Remina1),� Reina�Ozaki1),� Toyoaki�Murohara1)

1）Nagoya University Graduate school of Medicine

[Background]Glucagon (Gcg) is a peptide that regulate glucose metabolism. Gcg has pleiotropic effect and its acute administration promotes inotropic and chronotropic effects on myocardium. However, its pathophysiological role in cardiovascular system remains uncertain. [Methods]Cardiovascular changes in mice lacking Gcg and glucagon-like peptide-1 (GLP-1) by substitution of preproglucagon gene with GFP (Gcg-null) were evaluated with or without 1 month treatment of Gcg (Sup-Gcg; 2 mg/kg/d) or GLP-1 (Sup-GLP1; 100 µg/kg/d). The role of Gcg in heart failure were investigated by use of mouse model induced by transaortic constriction (TAC). [Results] Gcg-null exhibited hypertension [in systolic blood pressure (mmHg); 115±4 for Gcg-null and sBP 89±4 for wild]. Heart weight of Gcg-null was increased [(mg); 156.2±9.1 for Gcg-null and 130.7±4.5 for wild] and its systolic function was impaired [FS (%); 26.7±2.7 for Gcg-null and 42.4±4.8 for wild]. Interestingly , Gcg-nul l exhibited > 3 fold higher plasma catecholamine (CA) concentration with adrenal hypertrophy. Sup-Gcg reversed the CA surge and left ventricular (LV) dysfunction observed in Gcg-null, which were refractory to Sup-GLP1. TAC exhibited systolic LV dysfunction, which was reversed by Sup-Gcg. Notably, plasma CA elevation which was induced by TAC was >5 higher in Gcg-null than in wild. [Conclusion(s)] Gcg counteracts CA surge that causes hypertension and heart failure.

P18-1Novel compound heterozygous TMEM70 variants impaired the activity of ATP synthase of complex VKeiichi Hirono1),�Natsuhito�Nishio2),� Kei�Murayama5),�Yasushi�Okazaki3),�Akira�Ohtake4),�Fukiko�Ichida1)

1）University of Toyama, 2）Ishikawa Prefectural Central Hospital, 3）Juntendo University, 4）Saitama Medical University, 5）Chiba Children’s Hospital

TMEM70 is involved in the biogenesis of mitochondrial ATP synthase. Variants in the TMEM70 gene are the most frequent causes of human complex V defects of nuclear origin and its typical clinical features are cardiomiopathy, 3-methylglutaconic aciduria (3-MGA), and hypotonia. We identified novel compound heterozygous variants in the TMEM70 gene in Japanese patients who had hyperlactacidemia, metabolic acidosis and hyperalaninemia in his neonate. Developmental delay, undescended testicle, and left ventricular noncompaction were also observed. The urinary organic acids profile showed increased levels of 3-MGA. Complex V deficiency was confirmed by BN-PAGE/Western blotting analysis and ETC activity. Family screening revealed his old brother had same variants.We showed for the first time that the compound heterozygous variants in the TMEM70 gene were identified the Japanese patients. This report further may extend the clinical spectrum of TMEM70 gene defects.

P18-2HERPUD1 protects against ER stress- and oxidative stress-induced cardiomyocyte deathSoichiro Ikeda1),� Shouji�Matsushima1),�Kosuke�Okabe1),�Masataka�Ikeda1),�Tomomi�Ide1),�Hiroyuki�Tsutsui1)

1）Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, JapanBackgroundEndoplasmic reticulum (ER) stress is intimately involved in mitochondrial dysfunction and cardiac injury. HERPUD1, an ER membrane protein, is a component of ER associated degradation (ERAD) system which contributes to protein quality control in ER. However, the role of HERPUD1 in cardiac injury remains unclear. ObjectiveThe purpose of this study was to elucidate the role of HERPUD1 in mitochondrial dysfunction and cell death in cardiomyocytes induced by pathological stress including ER stress and oxidative stress.Methods and ResultsTunicamycin (TU:10ug/ml, 12h) induced upregulation of CHOP and Bip, markers of ER stress, and also increased HERPUD1 protein levels (6.5-fold, p<0.05) in neonatal rat ventricular myocytes (NRVMs). It increased cleaved cleaved caspase-3 (CC-3) and decreased mitochondrial function by oxygen consumption rate (OCR) and cardiomyocyte viability by cell titer blue assay in NRVMs. HERPUD1 was downregulated by small interfering RNA in NRVMs (NRVMs-H(-)). CHOP (1.2-fold, p<0.05) and Bip (1.4-fold, p<0.05) were further increased in NRVMs-H(-) compared with NRVMs after treatment with TU. Accordingly, CC-3 (1.3-fold, p<0.05) was increased and mitochondrial OCR (0.5-fold, p<0.05) and cell viability (0.7-fold, p<0.05) were decreased in NRVMs-H(-) after treatment with TU. To determine the effect of oxidative stress, NRVMs were treated with H2O2 (100μM, 2h). H2O2 increased HERPUD1 protein levels (1.3-fold, p<0.05) and decreased cell viability in NRVMs. CHOP, PERK, and CC-3 (1.2-, 1.9-, and 1.6-fold, respectively p<0.05) were increased and cell viability (0.8-fold, p<0.05) were further decreased in NRVMs-H(-) compared with NRVMs after treatment with H2O2. Myocardial infarction (MI) was created in c57BL/6 mice by ligating left coronary artery for in vivo analysis. HERPUD1 protein levels in non-infarcted left ventricle were increased 1 week after MI (1.5-fold, p<0.05), but it decreased to baseline level 4 weeks after MI, despite persistent elevation of CHOP and PERK during 4 weeks.Conclusions Downregulation of HERPUD1 exacerbates ER stress- and oxidative stress-induced cardiomyocyte death. Endogenous HERPUD1 might play a protective role in cardiac injury and dysfunction by attenuating ER stress, mitochondrial dysfunction, and cardiomyocyte death.

P18-3FKBP8 protects the heart by preventing accumulation of misfolded protein and ER-associated apoptosisTomofumi Misaka1),�Kinya�Otsu2),�Yasuchika�Takeishi1)

1）Fukushima Medical University, 2）King's College London

The accumulation of misfolded proteins and damaged organelles such as mitochondria in the heart is associated with heart failure. During the process to identify novel mitophagy receptors, we found FK506-binding protein 8 (FKBP8). The aim of this study was to determine the functional role of FKBP8 in the heart. Cardiac-specific FKBP8-deficient (Fkbp8 -/-) mice were generated. Fkbp8 -/- mice showed no cardiac phenotypes under baseline conditions. The Fkbp8-/- and control wild type littermates (Fkbp8+/+) mice were subjected to pressure overload by means of transverse aortic constriction (TAC). Fkbp8-/- mice showed left ventricular dysfunction and chamber dilatation with lung congestion 1 week after TAC. Deficiency of FKBP8 had no effect on mitophagy. The number of apoptotic cardiomyocytes was dramatically elevated in TAC-operated Fkbp8-/- hearts with an increase in cleaved caspase-12 and endoplasmic reticulum (ER) stress markers. Caspase-12 inhibition resulted in the attenuation of hydrogen peroxide-induced apoptotic cell death in FKBP8 knockdown H9c2 myocytes. Immunocytological and immunoprecipitation analyses indicated FKBP8 was localized to the ER and mitochondria in cardiomyocytes, interacting with heat shock protein 90. There was accumulation of misfolded protein aggregates in FKBP8 knockdown H9c2 myocytes and electron dense deposits in perinuclear region in TAC-operated Fkbp8-/- hearts. The data suggest FKBP8 plays a protective role against hemodynamic stress in the heart mediated via inhibition of the accumulation of misfolded proteins and ER-associated apoptosis.

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P18-7Supplementation of succinyl-CoA improves the mitochondrial complex II dysfunction in heart failureSatoshi Maekawa1),�Shingo�Takada1),�Takaaki�Furihata1),�Hideo�Nambu1),�Naoya�Kakutani1),�Shintaro�Kinugawa1),�Toshihisa�Anzai1)

1）Hokkaido universityBackground: Myocardial infarction (MI) leads to the development of heart failure (HF), which is the major cause of death of post-MI patients. The changes in left ventricular (LV) geometry and reduced ATP production based on mitochondrial dysfunction, i.e., remodeling, contribute to the development of depressed cardiac function in HF post-MI. The syntheses of heme and ketone body were reported to be increased in HF because a non-infarcted area of LV needs O2 and ATP. The key enzyme required for these syntheses is succinyl-CoA, which also plays an important role in providing succinate to mitochondrial complex II. However, the roles of succinyl-CoA and mitochondrial complex II in HF are unclear. Therefore, we attempted to determine whether succinyl-CoA affects the mitochondrial complex II function in HF. Methods & Results: We produced MIs in male C57BL/6J mice as an HF model by ligating the left anterior descending coronary artery. A sham operation was performed as the control. At 28 days post-surgery, echocardiography showed that cardiac function was significantly reduced and the heart and lung weights were significantly increased in the HF mice. LV of the mice were quickly harvested and mitochondria were isolated. Infarcted area of LV from MI mice was excluded. The protein levels in the mitochondria of both Oxct-1 (a key protein of ketone synthesis) and Alas (a key protein of heme synthesis) shown by immunoblotting were increased in the HF mitochondria. The succinyl-CoA level evaluated by LC/MS/MS was reduced in the HF mice. Since succinyl-CoA provides succinate to complex II, we evaluated complex II-linked respiration of isolated mitochondria. The complex II-linked respiration measured by high-resolution respirometry was significantly lowered in HF when succinate (the substrate for complex II) was saturated (Control vs. HF; 4,724 vs. 4,306 pmol/s/mg, p<0.05). This decline of respiration could be recovered by incubating the isolated mitochondria from HF mice with 0.1mM succinyl-CoA for 15 min (HF without succinyl-CoA vs. HF with 0.1mM succinyl-CoA; 3,291 vs. 4,037 pmol/s/mg, p<0.05). Conclusions: The supplementation of succinyl-CoA can improve the lowered mitochondrial complex II function in HF mouse heart. Succinyl-CoA may be a potential therapeutic target against mitochondrial dysfunction in HF patients.

P18-4Oxidant stress terminates protective signals in mitochondria by recruitment of DUSP5 and PHLPP-1.Wataru Ohwada1,2),�Masaya� Tanno1),� Toshiyuki� Yano1),�Takayuki�Miki1),�Atsushi�Kuno1),�Tatsuya�Sato1),�Tetsuji�Miura1)

1）Sapporo Medical University, 2）JCHO Sapporo Hokushin HospitalBackground: Fine-tuning of mitochondrial pro-survival and anti-survival signals determines cell death/survival. Here, we examined intra-mitochondrial localizations and phosphorylation of pro-survival kinases, and their regulation by protein phosphatases. Methods and Results: Mitochondria isolated by repetitive centrifugation from HEK293 or H9c2 cells underwent stepwise trypsin digestion; trypsin eliminated TOM20, an outer membrane (OM) protein, at 1 <g/ml and COX-IV, an inner membrane (IM) protein at 1,000 <g/ml. Immunoblotting revealed that protective treatment with IGF-1 (50 nmol/L) significantly increased mitochondrial GSK-3β, Akt and ERK with dominant localization being OM for GSK-3β and ERK, and IM for Akt. IGF-1 also increased the level of ser9-phospho-GSK-3β (by 42% and 52%), phospho-Akt (by 155% and 94%) and phospho-ERK (by 7% and 40%) in both OM and IM. Exposing cells to oxidant stress by using antimycin A (100 <mol/L, 30 min), a mitochondrial complex III inhibitor, significantly increased both DUSP5 and PHLPP-1, specific phosphatases for ERK and Akt, respectively, in the OM (by 86% and 52%, respectively), eliminating IGF-1-induced phosphorylation of Akt, ERK and GSK-3β. Experiments with mitochondria purified by percoll gradient centrifugation also confirmed the mitochondrial localization of DUSP5 and GSK-3β. Knockdown of PHLPP1 by siRNA significantly elevated phosphorylation level of both Akt and ERK in the mitochondria under treatment with antimycin A (by 370% and 190%). Similarly, the level of both phospho Akt and phospho ERK were significantly elevated (by 67% and 145%) by knockdown of DUSP5, suggesting a crosstalk between ERK and Akt in the mitochondria. Antimycin A significantly increased LDH release, an index of cell necrosis, from 7.6±1.3% in vehicle-treated cells to 14.0±2.5%, and siRNA-mediated knockdown of DUSP5 significantly suppressed LDH release (8.7±0.8%). Conclusion: Cytoprotective Ser9-phosphorylation of GSK-3β in mitochondria is mediated by Akt and ERK in the OM and IM, and recruitment of DUSP5 and PHLPP-1 to mitochondria contributes to ROS-induced termination of the protective signaling .

P18-5Inhibition of xanthine oxidase prevents skeletal muscle mitochondrial dysfunction in heart failureHideo Nambu1),� Shingo�Takada1),� Satoshi�Maekawa1),�Junichi� Matsumoto1), � Naoya� Kakutani1), � Shintaro�Kinugawa1),�Toshihisa�Anzai1)

1）Department of Cardiovascular Medicine, Faculty of Medicine and Graduate School of Medicine, Hokkaido UniversityIntroduction: Reduced exercise capacity in heart failure (HF) is closely associated with skeletal muscle abnormalities. We showed that mitochondrial function was impaired in skeletal muscle from HF mice and caused by increased reactive oxygen species (ROS). However, the pathogenic mechanism of mitochondrial dysfunction in association with increased ROS is unclear. Xanthine oxidase (XO) was reported to be an important mediator of ROS overproduction in ischemic tissue. Here we verified the hypothesis that skeletal muscle mitochondrial dysfunction in HF was initially caused by XO-derived ROS and prevented by its inhibition.Methods and Results: MI was created by ligating the left coronary artery, and a sham operation was also performed in male C57BL/6J mice. We first examined the time course after MI of XO activity, i.e. XO-derived ROS evaluated by hydrogen peroxide production, in skeletal muscle. The production of XO-derived ROS was significantly increased in the MI mice from 1 to 7 days post-surgery (initial phase), whereas it did not differ between both groups at 14 and 28 days (later phase). Second, we divided mice into three groups (n=8/group): sham+vehicle (SV), MI+vehicle (MV), and MI+febuxostat (an XO inhibitor, 5 mg/kg body weight/day; MF). Febuxostat or vehicle was administered by oral gavage at 1h and 24 h before and once-daily on days 1 to 7 post-surgery (initial phase). At day 28 post-surgery, exercise capacity as evaluated by treadmill testing was significantly decreased in the MV compared to the SV (Work; 17.5±0.8 vs. 23.0±1.5 J, P=0.02 vs. the SV), and this was preserved in the MF (23.3±1.1 J, P=0.02 vs. the MV) to the same extent as the SV in association with decreased XO-derived ROS without affecting the cardiac function and spontaneous physical activity. The mitochondrial respiration in skeletal muscle fibers shown by high-resolution respirometry were significantly decreased in the MV compared to the SV (O2 flux/wet weight; 165.1±14.6 vs. 233.1±11.2 pmol/(sec*mg), P=0.04 vs. the SV) and this preserved in the MF (282.6±2.4 pmol/(sec*mg), P=0.02 vs. the MV) to the same extent as the SV.Conclusion: XO inhibition during increased XO-derived ROS in the initial phase can prevent skeletal muscle mitochondrial dysfunction and exercise intolerance in HF after MI mice.

P18-6Cilnidipine improves heart failure after myocardial infarction by suppressing mitochondrial fissionKakeru Shimoda1,2),� Akiyuki� Nishimura1,2),� Tsukasa�Shimauchi1),� Tomohiro� Tanaka1),� Takuro� Numaga-Tomita1,2),�Motohiro�Nishida1,2,3)�1）Creative Research Group on Cardiocirculatory Dynamism, Exploratory Research Center on Life and Living Systems (ExCELLs), National Institutes of Natural Sciences, 2）Department of Physiological Sciences, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), 3）Department of Translational Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University

Disorders of mitochondrial dynamics is recognized as a key determinant of myocardial vulnerability after myocardial infarction (MI). Dynamin-related protein 1 (Drp1) accumulates at mitochondrial fission sites via actin cytoskeleton and induces mitochondrial fission, but the molecular mechanism is still unknown. Here, we identified Filamin A, an actin binding protein, as a new guanine nucleotide exchange factor of Drp1. Filamin A was upregulated in peri-infarct region of MI mouse hearts, and colocalized with Drp1 around myocardial mitochondria. Filamin A activated Drp1 in coordination with Actin, and this Filamin A-Actin-Drp1 complex mediated hypoxia- induced mitochondrial f ission and senescence in cardiomyocytes. Moreover, we found that cilnidipine, which is known as a dihydropyridine-derivative voltage-dependent L/N-type Ca2+ channel blocker, inhibited mitochondrial hyperfission by suppressing Drp1-Filamin A complex formation. In addition, administration of cilnidipine to mice ameliorated cardiac senescence and heart failure after MI. However, another dihydropyridine Ca2+ channel blocker, amlodipine, never ameliorated mitochondrial hyperfission and cardiac senescence. These results clearly suggest that cilnidipine improves chronic heart failure after MI by suppressing mitochondrial hyperfission in mice.

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INDEX

120

AAbe, Koki P08-5

Adachi, Yusuke P15-2

Adhikara, Imam Manggalya

P15-5

Akazawa, Hiroshi LS3

Aono, Jun P04-2

Aoyama, Junya SS2-4

Araki, Satoshi P11-6

Arima, Yuichiro GS-1, P13-8

Asano, Yoshihiro SS5-3

Ashida, Noboru P15-8

BBarinda, Agian Jeffilano P01-3

Bui, Thang Quoc P02-2

CChiba, Ayano P07-6

Cho, Kosai P13-4

DDatta, Partha Sarathi P11-1

De Vera, Julius Caesar

P06-5, P06-6

EEndo, Jin AS-5

Eschenhagen, Thomas Kl-2

FFujita, Kanna SS5-4

GGamble, Jennifer R SS4-1

Gao, Jingshan P16-3

Goto, Shinichi P03-6, P03-7

Gumdani, Lianita P03-2

HHaginiwa, Sho P07-4

Hashimoto, Ryota P04-3

Hashimoto, Youhei P02-5

Hata, Yoshihiro P08-8

Hatipoglu, Omer P02-7

Hausenloy, Derek SS3-1

Hayashi, Hiroki P10-7

Hayashi, Tomohiro P12-5

Hayashi, Yuka SS3-4

He, Yulong SS6-1

Heima, Daisuke P11-8

Higuchi, Yusuke P05-2

Hiraide, Takahiro P14-1

Hirono, Keiichi P18-1

Hirose, Masamichi SS1-2

Hirose, Masanori P04-5

Honda, Sakiko P01-8

Horie, Takahiro P13-6, P15-7

Horinaka, Shigeo LS4

Horitani, Keita P01-5

Horiuchi, Kiwamu P04-6

Hoshino, Atsushi SS3-3, GS-4

IIhara, Kensuke P16-7

Ikeda, Soichiro P18-2

Inazumi, Hideaki AS-2

Isagawa, Takayuki P04-1

Ishibashi, Tomohiko P15-6

Ishida, Toshifumi P12-2

Ishikawa, Hiroyuki P07-3

Ishikawa, Shohei P11-4

Iso, Tatsuya P12-6

Ito, Kei P16-5

Izumiya, Yasuhiro P11-2

JJiang, Jianming SS5-1

Jin, Huiling P17-5

Jin, Suk-Won SS7-1

KKadota, Shin P07-7

Kakino, Akemi P04-4

Kamatani, Yoichiro SS5-2

Kamihara, Takahiro P01-2

Kamimura, Daisuke SS4-4

Kamo, Takehiro P12-3

Kasamoto, Mnabu P07-8

Kashiwa, Asami P09-2

Katsumata, Yoshinori GS-3

Kawamata, Ryo P14-6

Kim, Hyo-Soo SS2-1

Kimura, Haruka P13-1

Kimura, Tomonari P13-7

Kimura, Toyokazu P17-3

Kimura, Wataru GS-5

Kira, Shintaro P10-2

Kishi, Takuya LS2

Koch, Walter J Kl-1

Kohjitani, Hirohiko P08-2

Kohno, Michiaki P05-6

Konishi, Takao P03-4

Kook, Hyun SS1-1

Kumazawa, Takuya

GS-2, P01-1, P05-8

Kuramoto, Yuki P09-6

Kuribayashi, Yuko P14-8

Kuwahara, Koichiro SS1-3

Kwon, Chulwon P06-4

LLi, Peili P16-6

Lino, Caroline Antunes P02-3

Liu, Qing P02-1

Lu, Weizhe P09-5

MMaejima, Yasuhiro SS7-4

Maekawa, Satoshi P18-7

Majima, Ryohei P02-6


121

Masumura, Yuki P05-7

Matsumoto, Kotaro AS-4

Mikami, Yoshinori P17-1

Minato, Takafumi P08-4

Misaka, Tomofumi P18-3

Miyagawa, Kazuya SS7-3

Miyamoto, Kazutaka AS-3

Miyashita, Yohei P09-3

Moki, Hikaru P08-3

Mori, Hiroyoshi P14-2

Mori, Yasuo SS8-2

Morooka, Nanami P07-5

Motoyama, Shinichiro P13-3

NNakagami, Hironori P08-1

Nakagawa, Hitoshi P06-3

Nakagawa, Osamu P07-1, P07-2

Nakamura, Kazufumi P14-4

Nakano, Tomoya P09-7

Nakaoka, Yoshikazu SS7-2

Nambu, Hideo P18-5

Nishimura, Kazuyuki P17-7

Nomura, Hidekimi P15-1

Nugroho, Dhite B P01-4

OOda, Sayaka P17-2

Oda, Tetsuro P05-5

Ogita, Hisakazu SS1-4

Ohwada, Wataru P18-4

Oimatsu, Yu P11-7

Oishi, Yumiko SS4-3

Okamoto, Hiroshi P03-5

Okazawa, Makoto P14-3

Okuno, Keisuke P11-3

Oniki, Takahiro P16-4

Osaki, Ayumu P15-3

Ow, Pei Chen Connie P12-8

PPini, Maria P10-8

RRyanto, Gusty Rizky Teguh P14-5

SSaburi, Makoto P10-1

Saga-Kamo, Akiko P12-4

Sakaguchi, Teruhiro P05-1

Sari, Nurmila P06-2

Sasano, Tetsuo SS8-3

Sato, Thomas N SS4-2

Satoh, Masahiro P09-8

Sawaki, Daigo P01-6

Setoyama, Daiki SS3-2

Shibamoto, Masato P10-6

Shimizu, Kana P05-4

Shimizu, Tatsuya SS2-2

Shimoda, Kakeru P18-6

Shin, Seungjae P02-4

Somsaart, Thatsaphone P03-1

Suematsu, Yasunori P04-8

Suetomi, Takeshi P10-3

Suganuma, Masashi LS5

Sunagawa, Yoichi P08-7

TTakakura, Nobuyuki SS6-2

Takeda, Norihiko LS1

Tanaka, Hideo P16-2

Tashiro, Kouhei P02-8

Tohyama, Shugo SS2-3

Tokunaga, Nobuyuki P15-4

Toshima, Taku P08-6

Tsuji, Yumika P11-5

Tsuruda, Toshihiro P01-7

UUchikado, Yoshihiro P04-7

Urayama, Kyoji P12-1

WWada, Naotoshi P13-5

Waddingham, Mark T P17-4

Wang, Wei SS8-1

Wardhana, Donytra Arby P14-7

Wu, Hua-Lin SS6-3

YYamaguchi, Noritaka P10-4

Yamamoto, Daiki P06-1

Yamamoto, Eri P03-3

Yamamoto, Yuta SS8-4

Yamamura, Satoru P05-3

Yamashiro, Yoshito SS6-4, P02-3

Yamazoe, Masahiro AS-1

Yang, Xiaoxi P09-4

Yasumura, Seiki P12-7

Yokokawa, Tetsuro P06-7

Yonebayashi, Saori P10-5

Yoshida, Naofumi P03-8

Yoshida, Yohko P13-2

Yoshii, Akira P17-6

Yuasa, Shinsuke LS1

ZZankov, Dimitar P09-1

Zhou, Xue P16-1

122

Sponsors ListDiamond

Takeda Pharmaceutical Company Limited.

PlatinumDaiichisankyo Co., Ltd.

Mitsubishi Tanabe Pharma Corporation.Nippon Boehringer Ingelheim Co ., Ltd.

Bayer Yakuhin, LtdOTSUKA Pharmaceutical Co.Ltd.

MSD K.K.Bristol-Myers Squibb

Pfizer Japan Inc.Astellas Pharma Inc.

Amgen Astellas BioPharma K.K.AstraZeneca K.K.

ONO PHARMACEUTICAL CO., LTD.

Goldemka TECHNOLOGIES JapanNIPPON SHINYAKU CO.,LTD.

Actelion Pharmaceuticals Japan Ltd.Johnson & Johnson K.K.GlaxoSmithKline K.K.Orange Science LLC

CANON MEDICAL SYSTEMS CORPORATIONPrimetech Corporation

NIHON KOHDEN CORPORATION

Silver Kowa Pharmaceutical Co. Ltd. Abbott Vascular Japan Co.,Ltd. Asahi Kasei ZOLL Medical Corporation. Eli Lilly Japan K.K. Medtronic Japan Co., Ltd. Chugai Pharmaceutical Co., Ltd. TERUMO CORPORATION Sumitomo Dainippon Pharma Co., Ltd. SANWA KAGAKU KENKYUSHO CO., LTD. Kyowa Hakko Kirin Co., Ltd. TOA EIYO LTD. SRL, Inc. MOCHIDA PHARMACEUTICAL CO., LTD. Boston Scientific Corporation Sanofi K.K. WAKENYAKU CO.,LTD. Siemens Healthcare K.K. Konishi Medical Instruments Co., Ltd. GlaxoSmithKline K.K. FUJI YAKUHIN CO., LTD. TEIJIN HOME HEALTHCARE LIMITED. TSUMURA & CO. TEIJIN PHARMA LIMITED KURITASHOTEN

AcknowledgmentTERUMO FOUNDATION for LIFE SCIENCES and ARTS

Nara Visitors Bureau

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September 2018 Dates 22SAT 23SUN · Award Session 24 ... screen-saver, virus scan and power ... ※Time would be delayed due to traffic jam